KR20220119611A - Method for producing natural killer cells and composition thereof - Google Patents

Abstract

A method for producing natural killer cells is disclosed. The method comprises isolating peripheral blood mononuclear cells (PBMCs) from a blood sample; isolating at least one of CD56+ cells and/or CD3-/CD56+ cells from the PBMCs; and co-culturing at least one of the CD56+ cells and/or the CD3-/CD56+ cells with the combination of feeder cells in the presence of the cytokine. The method may further comprise freezing and thawing CD56+ cells and/or CD3-/CD56+ cells. A composition for treating cancer is also disclosed. The composition comprises CD56+ natural killer cells and a cytokine produced by the disclosed method.

Description

Method for producing natural killer cells and compositions thereof

This application is US Provisional Application No. 63/062694, filed on August 7, 2020, and Korean Patent Application No., filed on November 29, 2019. KR-10-2019-0157727 claims priority, the contents of each of which are incorporated herein by reference in their entirety.

The present disclosure relates to the preparation, storage and/or per se of natural killer cells.

Natural killer cells (NK cells) are a type of innate immune cells, non-specifically killing cancer, recognizing and killing viruses, bacteria, etc., enzymes such as perforin, granzyme, or Fas-FasL interaction known to kill pathogens. In cancer patients, the reduction in cancer cell cytotoxicity of these NK cells is associated with lung cancer (Carrega P, et al., Cancer, 2008: 112: 863-875), liver cancer (Jinushi M, et al., J Hepatol., 2005: 43 1013-1020), breast cancer (Bauernhofer T, et al., Eur J Immunol., 2003: 33: 119-124), uterine cancer (Mocchegiani E., et al., Br j Cancer., 1999: 79: 244-) 250) and blood cancer (Tajima F., et al, Lekemia 1996: 10: 478-482), and has been reported to be associated with the development of various types of cancer.

The present application relates to methods for producing high-purity natural killer cells and to a cell therapy composition for cancer treatment comprising high-purity natural killer cells and cytokines. Any features, structures or steps disclosed herein may be substituted, combined, or omitted with any other features, structures or steps disclosed herein. Furthermore, for the purpose of summarizing the present disclosure, certain aspects, advantages, and features of the present invention have been described herein. It should be understood that some or all of these advantages may not necessarily be achieved in accordance with any particular embodiment of the inventions disclosed herein. No individual aspects of the present disclosure are essential or essential.

In some embodiments, methods of propagating natural killer cells in culture are disclosed. The method includes isolating CD56+ cells from a blood sample; co-culturing the isolated CD56+ cells in the presence of IL-21 {and feeder cells} for a first period; freezing the co-cultured CD56+ cells after the first period; thawing the frozen CD56+ cells; and co-culturing the thawed CD56+ cells in the presence of IL-21 (and feeder cells) for a second period.

A method of any of the embodiments provided herein and/or any method disclosed herein may include one or more of the following features. The method may further comprise storing the frozen CD56+ cells at a temperature of less than -100 °C. The method may further comprise storing the frozen CD56+ cells for at least one day prior to thawing. Isolated CD56+ cells can be co-cultured for 13-16 days (or 9-25 days) prior to freezing. Isolated CD56+ cells can be co-cultured with one or more irradiated feeder cells in the presence of IL-21. Thawed CD56+ cells can be co-cultured with one or more irradiated feeder cells in the presence of IL-21. One or more feeder cells include irradiated Jurkat cells, irradiated Epstein Barr virus-transformed lymphocyte continuous cell line (EBV-LCL) cells, K562 cells and PBMCs (e.g., including autologous PBMCs). It may be one or more selected from the group consisting of. CD56+ cells can be co-cultured at a ratio of CD56+ cells to feeder cells of about 1:1-100. In some embodiments, any feeder cell may be used for first, second, or first and second proliferation (eg, with Il-21). CD56+ cells can be co-cultured at a ratio of CD56+ cells to feeder cells of about 1:1, 1:2, 1:5, 1:10, 1:20, 1:30 or 1:100. In some embodiments, this ratio is for KL1/EBVLCL and for other feeder cells, for example, 1:1 to 1:10 may be used. IL-21 may be added at a concentration of 10-100 ng/mL during the first and/or second period. IL-21 may be added at a concentration of 20-80 ng/mL during the first and/or second period. IL-21 may be added at a concentration of 30-70 ng/mL during the first and/or second period. IL-21 may be added more than once during the first and/or second period.

In some embodiments, methods of propagating natural killer cells in culture are provided. The method comprises isolating CD56+ from a blood sample (eg, PBMC, fresh or frozen, umbilical cord blood, and/or blood from which CD56+, CD56+CD3- and CD3- cells have been isolated); co-culturing CD56+ cells with one or more feeder cells in the presence of IL-21; freezing the CD56+ cells; thawing the frozen CD56+ cells; and propagating the thawed CD56+ cells (again, with suitable feeder cells of any type).

The method of any embodiment and/or any method disclosed herein may include one or more of the following features. Freezing CD56+ cells can be performed at a temperature of less than -100 °C. The method may further comprise storing the frozen CD56+ cells for a period of more than one day and less than 10 years. CD56+ cells can be co-cultured for 13-16 (or 9-25) days prior to freezing. The one or more feeder cells are not limited in all embodiments and may be one or more selected from the group consisting of at least one of: irradiated Jurkat cells, irradiated Epstein Barr virus-transformed lymphocyte continuous cell line (EBV) -LCL) cells, K562 cells, mb15-k562, mb21-k562 feeder cells, HuT78, and/or PBMCs. CD56+ cells can be co-cultured at a ratio of CD56+ cells to feeder cells of about 1:1-100. IL-21 may be added at a concentration of 10-100 ng/mL. IL-21 may be added more than once. In some embodiments, NK cells can be used with any feeder cell type as long as IL-21 is used prior to freezing, and as long as it is thawed from freezing followed by a restimulation process.

In some embodiments, methods of increasing the cytotoxicity of natural killer cells are disclosed. The method comprises providing said natural killer cells; freezing the natural killer cells; thawing the frozen natural killer cells; and co-culturing the thawed natural killer cells with one or more feeder cells in the presence of IL-21. Optionally, natural killer cells can be co-cultured (proliferated) with feeder cells and IL-21 prior to freezing the natural killer cells.

Any method in any of the preceding paragraphs and/or any method disclosed herein may include one or more of the following features. The method may further comprise the step of storing the frozen natural killer cells at a temperature of less than -100 °C. The method may further comprise the step of storing the frozen natural killer cells for at least one day before thawing. One or more feeder cells may be selected from the irradiated Jurkat cell, the irradiated Epstein-Barr virus-transformed lymphocyte continuous cell line, irradiated Jurkat cells, the irradiated Epstein-Barr virus-transformed lymphocyte continuous cell line (EBV- LCL) cells, K562 cells, mb15-k562, mb21-k562 feeder cells, HuT78, and/or PBMCs. Thawed natural killer cells can be co-cultured at a ratio of about 1:1-100 CD56+ cells to feeder cells. IL-21 may be added at a concentration of 10-100 ng/mL. IL-21 may be added more than once.

In some embodiments, a method of treating a subject is disclosed. The method comprises collecting CD56+ cells from a subject; co-culturing CD56+ cells with one or more feeder cells in the presence of IL-21; freezing the co-cultured CD56+ cells for at least one day; thawing the frozen CD56+ cells; propagating thawed CD56+ cells; and administering the proliferated CD56+ cells to the subject, wherein the cytotoxicity of the cells obtained from the second proliferation is at least X% of the cytotoxicity of the co-cultured CD56+ cells prior to freezing.

Any method in any of the preceding paragraphs and/or any method disclosed herein may include one or more of the following features. The method may further comprise storing the frozen CD56+ cells at a temperature of less than -100 °C. The method may further comprise storing the frozen CD56+ cells for at least one day prior to thawing. Isolated CD56+ cells can be co-cultured for 13-16 (or 9-25) days prior to freezing. Proliferating the thawed CD56+ cells may include co-culturing the thawed CD56+ with one or more irradiated feeder cells in the presence of IL-21. One or more feeder cells are irradiated Jurkat cells, irradiated Epstein Barr virus-transformed lymphocyte continuous cell line (EBV-LCL) cells, K562 cells, mb15-k562, mb21-k562 feeder cells, HuT78 , and/or one or more cells selected from the group consisting of PBMCs. CD56+ cells can be co-cultured at a ratio of CD56+ cells to feeder cells of about 1:1-100. IL-21 may be added at a concentration of 10-100 ng/mL during the first and/or second period. IL-21 may be added more than once during the first and/or second period.

In some embodiments, a composition is provided. The composition comprises an effective amount of CD56+ cells derived from peripheral blood mononuclear cells (PBMCs) from a patient. CD56+ cells can be obtained by isolating peripheral blood mononuclear cells (PBMCs) from a blood sample; isolating CD56+ cells from PBMCs; co-culturing CD56+ cells with one or more feeder cells in the presence of one or more cytokines; freezing the CD56+ cells; thawing the frozen CD56+ cells; and co-culturing the thawed CD56+ cells with one or more feeder cells in the presence of one or more cytokines; prepared through

In some embodiments, a cell composition is disclosed. The cell composition comprises: an effective amount of CD56+ cells derived from peripheral blood mononuclear cells (PBMCs) from a patient; IL-2; and IL-21.

In some embodiments, a composition is disclosed. The composition comprises a first population of CD56+ cells derived from peripheral blood mononuclear cells (PBMCs); ice; IL-2; and IL-21. When thawed, the CD56+ cells have at least 80% cytotoxicity of the second population of CD56+ cells, the second population of CD56+ cells being unfrozen.

In some embodiments, methods of propagating natural killer cells in culture are provided. The method comprises: isolating CD56+ cells from a blood sample; co-culturing the isolated CD56+ cells in the presence of IL-21 for a first period; freezing the co-cultured CD56+ cells after the first period; thawing the frozen CD56+ cells; and co-culturing the thawed CD56+ cells in the presence of IL-21 for a second period.

In some embodiments, methods of propagating natural killer cells in culture are provided. The method comprises: isolating CD56+ from a blood sample; co-culturing CD56+ cells with one or more feeder cells in the presence of IL-21; freezing the CD56+ cells; thawing the frozen CD56+ cells; and propagating the thawed CD56+ cells.

In some embodiments, a method of increasing the cytotoxicity of natural killer cells is provided, comprising the steps of providing said natural killer cells; freezing the natural killer cells; thawing the frozen natural killer cells; and co-culturing the thawed natural killer cells with one or more feeder cells in the presence of IL-21.

In some embodiments, a method of treating a subject is provided, comprising: collecting CD56+ cells from the subject; co-culturing CD56+ cells with one or more feeder cells in the presence of IL-21; freezing the co-cultured CD56+ cells for at least one day; thawing the frozen CD56+ cells; propagating thawed CD56+ cells; and administering the proliferated CD56+ cells to the subject, wherein the cytotoxicity of the cells obtained from the second proliferation is at least 80% of the cytotoxicity of the co-cultured CD56+ prior to freezing.

In some embodiments, a composition is provided comprising: an effective amount of CD56+ cells derived from peripheral blood mononuclear cells (PBMCs) from a patient, the CD56+ cells being prepared by: peripheral from a blood sample isolating blood mononuclear cells (PBMCs); isolating CD56+ cells from PBMCs; co-culturing CD56+ cells with one or more feeder cells in the presence of one or more cytokines; freezing the CD56+ cells; thawing the frozen CD56+ cells; and co-culturing the thawed CD56+ cells with one or more feeder cells in the presence of one or more cytokines.

In some embodiments, a cell composition is provided comprising: an effective amount of CD56+ cells derived from peripheral blood mononuclear cells (PBMCs) from a patient; IL-2; and IL-21.

In some embodiments, a composition is provided comprising: a first population of CD56+ cells derived from peripheral blood mononuclear cells (PBMCs); ice; and IL-2, IL-21. When thawed, the CD56+ cells were cytotoxic to at least 80% of the second population of CD56+ cells and the second population of CD56+ cells was not frozen.

In some embodiments, a method of propagating natural killer cells in culture is provided, comprising: providing PBMCs; co-culturing the PBMCs in the presence of IL-21 for a first period; freezing the co-cultured PBMCs after the first period; thawing frozen PBMCs; and co-culturing the thawed PBMCs in the presence of IL-21 for a second period.

In some embodiments, the composition comprises: IL-2; 5-10% DMSO; 90-95% FBS; and NK cells, optionally CD56+ cells. In some embodiments, it further comprises a CryoStor solution. In some embodiments, the composition is for frozen cells prior to re-proliferation.

In some embodiments, the composition comprises IL-2; 5-10% DMSO; 80-95 % Hartman solution; 1-10% human serum albumin; and NK cells. In some embodiments, it further comprises a CryoStor solution. In some embodiments, the composition is for cells frozen prior to injection.

Methods have been developed and provided herein to help NK cells undergo and recover from cryopreservation. It has been found that CD56+ cells can be successfully propagated after freezing and thawing when CD56+ cells are co-cultured with feeder cells in the presence of IL-21 initially (during initial proliferation). By using IL-21, high-purity CD56+ NK cells can be generated, and surprisingly, they retain a particularly high amount of cytotoxicity. Moreover, after the NK cells have been thawed, they can be further expanded (without IL-21, or even more advantageously, in the presence of additional IL-21). Thus, IL-21 in the pre-freezing process (eg first propagation) may allow good re-proliferation later. In addition, the resulting product, even after being propagated twice and frozen once, retains a surprisingly high amount of cytotoxicity, which is more pronounced when IL-21 is used for the first as well as the second growth. is strengthened In some embodiments, this freezing and re-growth process may be repeated multiple times (each time, optionally with another step of IL-21 during re-proliferation).

In some embodiments, methods of propagating natural killer cells in culture are provided. The method includes providing PBMCs; co-culturing the PBMCs in the presence of IL-21 for a first period; freezing the co-cultured PBMCs after the first period; thawing the frozen PBMCs; and co-culturing the thawed PBMCs in the presence of IL-21 for a second period. In some embodiments, the ratio of PBMCs to feeder cells is 1:0.5:0.5. In some embodiments, the ratio may be about 1:0.5:0.5 to 1:10:10 for PBMC (eg, as an alternative to CD56+). In some embodiments, for CD56+ cells: the ratio is multiplied, eg, by 10 or 20 (eg, CD56+ cells to feeder cells 1:1-100). In some embodiments, proliferation occurs from CD56+ or CD56+/CD3- cells.

According to some embodiments, a method of producing high-purity NK cells may comprise: administering cells selected from CD56+ cells and/or CD3-/CD56+ cells to a first cytokine, e.g., IL-21 co-culturing with feeder cells in the presence of ("first culture step" or "first proliferation step"); freezing the co-cultured cells (“freezing step”); thawing the frozen cells ("thawing step"); and co-culturing the thawed cells with the added feeder cells, optionally with more IL-21 (“second culture phase” or “second proliferation phase”). Each step is described in more detail herein. CD3-/CD56+ cells produced according to the disclosed method exhibit higher purity and higher anti-cancer activity, as well as different surface markers or activated receptors, e.g., CD16, CD25, CD27, CD28, CD69 , CD94/NKG2C, CD94/NKG2E, CD266, CD244, NKG2D, KIR2S, KIR3S, Ly94D, NCR, IFN-a, IFN-b, CXCR3, CXCR4, CX3CR1 and CD577. can represent

As used herein, a “step” is part of a process and one “step” need not be completed before the next “step” begins. Unless otherwise specified, steps may be provided concurrently or in overlapping periods, where appropriate. Of course, if one stage occurs before the event (eg freezing) and the other stage occurs after the same event, there is no overlap (eg, first IL-21 culture and second IL-21 culture).

As used herein, the term “CD56+ cells (CD56+ cells)” may be used interchangeably with “CD56+ NK cells” or “CD56+ natural killer cells”, and the term “CD3-/CD56+ cells (CD3-/ CD56+ cells)" may be used interchangeably with "CD3-/CD56+ NK cells". CD56+ cells or CD3-/CD56+ cells may comprise cells expressing cell surface CD56 glycoprotein, or further, cells in which CD3 glycoprotein is not expressed while CD56 glycoprotein is expressed. Even the same type of immune cell may have different functions because the type and expression rate of CD attached to the cell surface are different.

In some embodiments, CD56+ cells or CD3-/CD56+ cells are obtained through the following steps: isolating peripheral blood mononuclear cells (PBMCs) from a blood sample ("first isolation step"); isolating cells selected from the group consisting of CD56+ cells and CD3-/CD56+ cells from peripheral blood mononuclear cells (“second isolation step”).

As used herein, "blood sample" may be whole blood of peripheral blood or white blood cells isolated from peripheral blood using leukapheresis, but is not limited thereto. In addition, the peripheral blood may be collected from a normal person, a patient at risk of cancer, or a cancer patient, but the source of the peripheral blood is not limited thereto.

As used herein, the term "leukapheresis" may refer to a method of selectively removing (separating) leukocytes from the collected blood and then transfusing the blood back to the patient, and in some embodiments, by the method The isolated leukocytes can be used without additional methods such as the Ficoll-Hypaque density gradient method.

As used herein, the term "peripheral blood mononuclear cell" may be used interchangeably with "PBMC", "monocyte", and is generally used for anti-cancer immunotherapy isolated from peripheral blood. It may mean a mononuclear cell. Peripheral blood mononuclear cells can be obtained from human blood collected using known methods such as the Ficoll-Hypaque density gradient method.

In some embodiments, peripheral blood mononuclear cells may be autologous, but allogeneic peripheral blood mononuclear cells may also be used to produce high-purity NK cells for anti-cancer immunotherapy according to the methods described herein. can Moreover, in some embodiments, peripheral blood mononuclear cells may be obtained from a normal person, but peripheral blood mononuclear cells may also be obtained from a patient at risk for cancer and/or a cancer patient.

In some embodiments, the second separation step for isolating CD56+ natural killer cells from the blood sample uses at least one selected from the group consisting of CD56 microbeads and CD3 microbeads, or CliniMACS, Separation may be performed by a method of separation using equipment such as a flow cytometry cell sorter, MACS Separator, or a magnetic sorting system.

For example, a separation method using CD56 microbeads and/or CD3 microbeads is performed by adding CD56 microbeads to PBMCs and then removing non-specific binding, or by adding CD3 microbeads to PBMCs. After removal of specific binding, non-specific binding may be removed by adding CD56 microbeads again. In some examples, T cells or other non-natural killer cells can be eliminated by isolating CD56+ cells and/or CD3-/CD56+ cells from PBMCs.

As used herein, the term “feeder cell” may refer to a cell that does not divide and proliferate but has metabolic activity, thereby helping the proliferation of target cells by producing various metabolites.

In some embodiments, the feeder cells are irradiated Jurkat cells, irradiated Epstein-Barr virus transformed lymphocyte continuous cell line (EBV-LCL) cells, PBMCs, HFWT, RPMI 1866, Daudi, MM- 170, K562 or K562 targeting and genetically modified cells (eg, K562-mbIL-15-41BB ligand) may be at least one selected from the group consisting of. For example, in one embodiment, the feeder cells may be irradiated Jurkat cells and EBV-LCL cells. In some embodiments, when NK cells are first proliferated in the presence of IL-21 and then frozen, thawed, and then re-proliferated, any feeder cell type, as provided herein, allows for re-proliferation. this can be used

As used herein, the term "Jurkat cell" or "Jurkat cell line" is a hematological cancer {immortalized acute T-cell leukemia, developed by Dr. Arthur Weiss of the University of California at San Francisco. acute T cell leukemia)} cell line. Jurkat cells, capable of expressing various chemokine receptors and producing IL-2, are a possible source of feeder cells for anti-cancer immunotherapy because MHC class I, a natural killer cell activation inhibitor, is highly expressed on the cell surface. It was not generally considered as a candidate. Jurkat cells can be obtained from ATCC (ATCC TIB-152).

As used herein, the term “EBV-LCL cell” or “EBV-LCL cell line” refers to an Epstein-Barr virus-transformed lymphocyte continuous cell line (EBV-LCL). (DM Koelle et al., J Clin Invest, 1993: 91: 961-968), a B cell line prepared by in vitro infection of human B cells with Epstein-Barr virus. EBV-LCL cells can be directly prepared and used in a general laboratory by adding cyclosporin A during EBV infection in PBMCs. In some embodiments, EBV-LCL cells can be prepared by the following steps. Add 30 x 10 ⁶ PBMCs to 9 mL of culture medium, add the mixture to a T 25 culture flask, and then add 9 mL of EBV supernatant. Add 80 µL of cyclosporin A (50 µg/mL) and incubate at 37 °C. After 7 days of culture, remove half of the supernatant, add fresh culture medium, and then add 40 µL of cyclosporin A. The same procedure can be repeated once every 7 days until the 28th day of culture. The cell line can be used after 28 days of culture, and from this point on, the cell line can be cultured without adding cyclosporin A to the culture medium.

Jurkat cells and EBV-LCL cells can be used as feeder cells after irradiation.

In some embodiments, prior to administration, the method may further comprise freezing the proliferated cells a second time in an injection-ready solution.

In some embodiments, the irradiated Jurkat cells and the irradiated EBV-LCL cells are 1:0.1-5, 1:0.1-4, 1:0.1-3, 1:0.1-2, 1:0.1-1.5, 1:0.5-1.5, 1:0.75-1.25, 0.1-5:1, 0.1-4:1, 0.1-3:1, 0.1-2:1, 0.1-1.5:1, 0.5-1.5:1, or 0.75 It can be included in a content ratio of -1.25:1. For example, irradiated Jurkat cells and irradiated EBV-LCL cells may be included in a content ratio of 1:1.

In some embodiments, the irradiated Jurkat cells and the irradiated EBV-LCL cells are 50-500, 50-400, 50-300, 50-200, 50-150, 70-130, 80-120 or 90- It can be obtained by treatment with 110 Gy radiation. For example, irradiated Jurkat cells and/or irradiated EBV-LCL cells can be obtained by treating Jurkat cells and/or EBV-LCL cells with 100 Gy radiation.

As used herein, the term "cytokine" may be used interchangeably with "a first cytokine" or "a second cytokine", which may be used to induce differentiation of peripheral blood mononuclear cells into NK cells. It may mean an immunoactive compound capable of In some embodiments, the cytokine is IL-21 (for the first and second proliferation, and any further phases of proliferation).

As used herein, the terms "co-culture" and "expansion" are interchangeable and refer to the proliferation of a population of cells in which NK cells have been cultured. The term “re-expansion” indicates that one stage of co-culture or proliferation has already occurred for NK cells. In some embodiments, co-culture or proliferation will occur in the presence of feeder cells such as IL-21 and cytokines. In some contexts herein, the term “culture” is used as an abbreviation for “co-culture”.

In some embodiments, the cytokine is interleukin-2 (IL-2), IL-15, IL-21, FMS-like tyrosine kinase 3 ligand (Flt3-L), stem cell factor (SCF), IL-7, IL -18, IL-4, type I interferon, granulocyte-macrophage colony-stimulating factor (GM-CSF) and insulin-like growth factor 1 (IGF 1), but are not limited thereto.

In some embodiments, the first cytokine is IL-2, IL-21, IL-15, FMS-like tyrosine kinase 3 ligand (Flt3-L), stem cell factor (SCF), IL-7, IL-18, IL-4, type I interferon, GM-CSF, insulin-like growth factor 1 (IGF 1), or a combination thereof. In some embodiments, the second cytokine is IL-2, IL-21, IL-15, FMS-like tyrosine kinase 3 ligand (Flt3-L), stem cell factor (SCF), IL-7, IL-18, IL-4, type I interferon, GM-CSF, insulin-like growth factor 1 (IGF 1), or a combination thereof. For example, the second cytokine may be IL-21. In some embodiments, one or more cytokines may be present across one or more steps provided herein. In some embodiments, both Il-21 and IL-2 are present in at least one of a first and a second proliferation phase (or any subsequent phase thereof). In addition, since the various embodiments provided herein involve the repeated use of a cytokine (eg, IL-21) in each of the various stages of proliferation, such cytokine can be used for the first time (or during the first stage or stage). and may be used a second (eg, during a re-proliferation phase). Thus, in some embodiments, a first cytokine (eg IL-21) may be used in both the first and second steps of co-culture. It may also alternatively be referred to as, for example, a first cytokine and a second cytokine, both of which are IL-21.

Methods for producing NK cells

1 is a flow chart showing some methods of propagating NK cells using various exemplary feeder cells. In some embodiments, CD56+ cells or CD3-/CD56+ cells are proliferated by co-culturing with feeder cells in the presence of IL-21. Feeder cells can be any type of feeder cell, e.g., Jurkat cells and EBV-LCL cells (“Type 1”), K562 cells (“Type 2”) or PBMCs (“Type 3”). have. In some embodiments, the cells are collected at about day 17 of culture (or anywhere on days 16-21 or 9-25), and the resulting cells will be referred to as "IL21+" above or elsewhere herein. can This cell proliferation process without cryopreservation or a second culturing step may be referred to as the "native process" above or elsewhere herein. In some embodiments, the cells are collected and cryopreserved at about 14 days (or anywhere from 14-18 or 9-25 days). Cultivation prior to cryopreservation may be referred to above or elsewhere herein as "first culturing step" or "first propagation step" or "first co-culturing step". Cryopreserved cells are thawed and re-proliferated by co-culture with feeder cells in the presence of IL-21 (“IL-21+/+”) or in the absence of IL-21 (“IL-21+/-”). can be This second propagation process may be referred to as a “second culturing step” or “re-stimulation process” or a “second co-culture step” or a second propagation step. Cells can be collected around 17 days of culture (or anywhere from 16-21 days or 9-25 days). In some embodiments, additional re-proliferation or re-stimulation steps or cycles may be performed. 14B , in some embodiments there may be a first stimulation followed by two or more re-stimulation steps.

In some embodiments, CD56+ cells or CD3-/CD56+ cells are proliferated by co-culturing with feeder cells in the absence of IL-21. In general, this applies to culturing steps after the initial culturing step with IL-21. Feeder cells are NK cells, including, for example, Jurkat cells and EBV-LCL cells (“Type 1”), K562 cells (“Type 2”) or PBMCs (“Type 3”). It may be any type of feeder cell for In some embodiments, the cells are harvested at about day 17 (or day 16-21) of culture, and the resulting cells may be referred to as “IL21-” above or elsewhere herein. In some embodiments, the cells are collected and cryopreserved at about 14 days (or 14-18 days). Cryopreserved cells are thawed and proliferated again by co-culture with feeder cells in the presence of IL-21 (“IL-21-/+”) or in the absence of IL-21 (“IL-21-/-”). can be

This second propagation process may be referred to as a 'second culture step' or a 're-stimulation process'. Cells can be collected at about 17 days of culture (or 16-21 or 9-25 days). As detailed herein, the use of IL-21 in the first proliferation, optionally along with additional IL-21 (eg, further benefits of improved cytotoxicity), is the freezing and thawing of NK cells. and subsequent superior propagation.

First culture (proliferation or co-culture) step

The first culturing step may include adding cytokines one or more times between culture days 0-6. One or more cytokines may be used (eg IL-2 may also be used). For example, the first culturing step may include adding one or both of the cytokines once each on days 0 and 3 of culture.

When co-culturing with feeder cells and the first cytokine, culturing with additional addition of another cytokine one or more times for 0-6 days may exhibit excellent proliferation and/or anti-cancer activity. In some embodiments, culturing with the addition of feeder cells and additional cytokines for 6 days in a 14-day cycle may exhibit superior proliferative and/or anti-cancer activity. In some embodiments, IL-21 is used at least once, optionally before and after freezing. In some embodiments IL-2 may be included as an additional cytokine.

In some embodiments, the first cytokine (eg, IL-21) is 10-1,000, 10-500, 10-100, 20-100, 30-100, 40-100, 50-100, or 10- It can be used at a concentration of 50 ng/mL. In some embodiments, the additional cytokine is 50-1,000, 50-900, 50-800, 50-700, 50-600, 50-550, 100-550, 150-550, 200-550, 250-550, 300 Concentrations of -550, 350-550, 400-550, or 450-550 IU/mL may be used. In some embodiments, the concentration is about 50 ng/ml.

Existing methods for proliferating NK cells use high concentrations of various cytokines. Conversely, in some embodiments of the methods of propagating NK cells described herein, NK cells in high yield and high purity can be propagated using only a low concentration of one cytokine.

In some embodiments, the co-culturing (culturing, propagation (including re-proliferation)) comprises 1:1- culturing peripheral blood mononuclear cells and feeder cells (eg, Jurkat cells and EBV-LCL cells). 100, 1:1-90, 1:1-80, 1:1-70, 1:10-65, 1:20-65, 1:30-65, 1:40-65, 1:50-65 or It can be carried out by mixing in a ratio of 1:55-65. In some embodiments, co-culture can be performed by including peripheral blood mononuclear cells and feeder cells (eg, Jurkat cells and EBV-LCL cells) in various mixing ratios. In some embodiments, the ratio may be about 1:0.5:0.5 to 1:10:10 for PBMC (eg, as an alternative to CD56+). In some embodiments, for CD56+ cells: the ratio is multiplied by, for example, 10 or 20 (eg, 1:1-100 of CD56+ cells to feeder cells).

The co-culture may be performed in a medium, and as the medium, an appropriate medium generally used for induction and proliferation of peripheral blood mononuclear cells into NK cells in the art may be used without limitation. For example, RPMI-1640, DMEM, x-vivo10, x-vivo20 or cellgro SCGM medium can be used as such medium. In addition, culture conditions such as temperature may follow appropriate culture conditions of peripheral blood mononuclear cells known in the art.

In some embodiments, the first culturing step is 0-45, 0-42, 0-40, 0-30, 0-20, 0-19, 0-18, 0-17, 0-16, 0-15 or It can be performed for 0-14 days.

freezing stage

Natural killer cells cultured and provided in the first culture step may be recovered and suspended in a medium and then frozen and cryopreserved. In some embodiments, the medium may comprise FBS and/or DMSO. For example, the medium may comprise 90% FBS and 10% DMSO, or 90-95% FBS and 5-10% DMSO. In some embodiments, other acceptable cryo-preservatives such as CryoStor solutions (CS10, CS5) or the like or other ingredients such as sucrose or glycerol may be included. In some embodiments, suitable preservatives include DMSO, glycerol, ethylene glycol, sucrose, trehalose, dextrose, polyvinylpyrrolidone, and the like. In some embodiments, IL-2 and/or Human Serum Albumin may be present.

In some embodiments, cryopreservation involves transferring the provided natural killer cells to a cryopreservation container with isopropyl alcohol, freezing the natural killer cells in the cryopreservation container overnight in a cryogenic freezer, and preserving the natural killer cells at -192 °C or less. may include In some embodiments, the frozen natural killer cells are -10 °C or lower, -20 °C or lower, -50 °C or lower, -70 °C or lower, -100 °C or lower, -150 °C or lower, -192 °C or lower, or -200 °C or lower. It can be preserved as frozen below. In some embodiments, the frozen natural killer cells are at least 1 day, at least 2 days, at least 3 days, at least 7 days, at least 14 days, at least 30 days, including any range between any two of the following values: It may be preserved for at least 60 days, or at least 180 days. In some embodiments, the temperature is between -135 °C and -196 °C. In some embodiments, the cells are stored for 0.5, 1, 2, 3, 4, or 5 years, inclusive of any range between any two of the following values.

In some embodiments, provided natural killer cells may be cooled and/or frozen using a controlled rate freezer (CRF). In some embodiments, frozen natural killer cells can be preserved under liquid nitrogen.

In some embodiments, provided natural killer cells may be cooled and/or frozen using a controlled rate freezer (CRF). In some embodiments, this can be done at a slow rate (eg, 1-8 hours, eg, 1, 2, 3, 4, 5, 6, 7, or 8 hours or more). Vials of NK cells can be manually frozen slowly using isopropyl alcohol stored in cryo-containers (eg, Nalgene Mr. Frosty) and stored at -70°C overnight. The next day the cells are transferred to liquid nitrogen (LN2).

thawing phase

Frozen natural killer cells can be thawed after cryopreservation using an appropriate method. In some embodiments, cryopreserved natural killer cells can be thawed using a water bath, eg, at 37°C. In some embodiments, frozen natural killer cells can be thawed for at least 1 hour, at least 2 hours, at least 5 hours, or for 10 hours. In some embodiments, the process is performed in a water or bead bath. In some embodiments, the thawing process is performed as soon as possible or immediately after being brought from a frozen state (eg, liquid nitrogen).

In some embodiments, frozen natural killer cells can be thawed in a 37°C water bath within 10 minutes, wherein the frozen vial or bag can be shaken to speed up the thawing process. In some embodiments, the cells are also subjected to an instrument such as a heat block, an automatic cell thawing device for vials (eg, ThawStar, Biocision), or a thawing device for bags (eg, VIA Thaw, GE Healthcare). It can be thawed using

a second culturing (second co-culturing, second propagation, or re-proliferation, or any subsequent culturing step) step

Since the cells are initially treated with IL-21 in the first culture step, a second proliferation step is possible. Thus, the method may include not only one propagation step, but two propagation steps (eg, one or more re-proliferation steps). Preferably, the second growth phase occurs after the sample is frozen, stored for a period of time, and then thawed.

During the second culturing step, thawed natural killer cells may be cultured by adding feeder cells one or more times.

In some embodiments, feeder cells may be added one or more times during a 14 day cycle (or 9-25 day cycle) of culture.

In some embodiments, culturing with the addition of feeder cells one or more times during a 14-day cycle not only exhibits superior proliferative and/or anti-cancer activity, but also maintains sustained cell growth even after freezing and thawing, so that natural killer cells are clinically to be produced in sufficient quantities for use in

In some embodiments, the second culturing step can include adding a cytokine from the second step (eg, additional IL-21 or other cytokines in addition to IL-21).

In some embodiments, the second culturing step may comprise adding the cytokine of the subsequent step one or more times during days 0-6 of culturing.

Descriptions of cytokines elsewhere herein can be applied to cytokines for the second culturing step.

For example, in some embodiments, the second cytokine is at a concentration of 10-1,000, 10-500, 10-100, 20-100, 30-100, 40-100, 50-100, or 10-50 ng/mL. concentrations and/or additional cytokines may be 50-1,000, 50-900, 50-800, 50-700, 50-600, 50-550, 100-550, 150-550, 200-550, 250- 550, 300-550, 350-550, 400-550, or 450-550 IU/mL. The cytokine used for the second proliferation is preferably IL-21.

In some embodiments, the composition is one of the cells frozen prior to re-proliferation, which may comprise: IL-2; 5-10% DMSO; 90-95% FBS; and NK cells, optionally CD56+ cells. In some embodiments, the composition is a frozen solid. In some embodiments, the NK cells are at least 90% of the cell population of the composition. In some embodiments, it further comprises a CryoStor solution. In some embodiments, the composition is for frozen cells prior to re-proliferation.

In some embodiments, the composition is for cells frozen prior to re-proliferation, which may comprise: IL-2; 5-10% DMSO; 80-95% Hartmann's solution; 1-10% human serum albumin; and NK cells. In some embodiments, it further comprises a CryoStor solution. In some embodiments, the composition is for cells frozen prior to injection.

In some embodiments, a method of propagating natural killer cells in culture comprises isolating CD56+ cells from a blood sample; co-culturing the isolated CD56+ cells in the presence of IL-21 for a first period; freezing the co-cultured CD56+ cells after the first period; thawing the frozen CD56+ cells; and co-culturing the thawed CD56+ cells in the presence of IL-21 for a second period.

In some embodiments, the method can further comprise storing the frozen CD56+ cells at a temperature of less than -100 °C. In some embodiments, the frozen CD56+ cells are stored at a temperature of -10 °C or less, -20 °C or less, -50 °C or less, -70 °C or less, -150 °C or less, -192 °C or less, or -200 °C or less. can In some embodiments, frozen CD56+ cells may be stored for at least one day prior to thawing. In some embodiments, the frozen CD56+ cells are at least 1 day, at least 2 days, at least 3 days, at least 7 days, at least 14 days, at least 30 days, including any range between any two of the following values: , for at least 60 days, or for at least 180 days. In some embodiments, cells can be frozen as long as they are viable when thawed.

In some embodiments, isolated CD56+ cells can be co-cultured for 13-16 days prior to freezing. For example, isolated CD56+ cells can be co-cultured for 14 or 15 days prior to freezing. In some embodiments, co-culture or propagation may proceed for an appropriate amount of time. In some embodiments, co-culturing or propagation (including re-proliferation) is 9-25 days, e.g., 10-24, 11-23, 13-22, 14-21, 14-18, 14-16 days. , and so on. These time frames can be applied to any of the proliferation and/or re-proliferation periods provided herein (including examples for other cells).

In some embodiments, isolated CD56+ cells can be co-cultured with one or more irradiated feeder cells in the presence of IL-21. In some embodiments, thawed CD56+ cells can be co-cultured with one or more irradiated feeder cells in the presence of IL-21. One or more feeder cells are irradiated Jurkat cells, irradiated Epstein-Barr virus transformed lymphocyte continuous cell line (EBV-LCL) cells, K562 cells, mb15-k562, mb21-k562 feeder cells, HuT78 And/or may include one or more selected from the group consisting of PBMC, but is not limited thereto. In some embodiments, propagation is performed with PBMC, CD56+ and/or CD56+CD3- cells. In some embodiments, CD56+ cells may be co-cultured at a ratio of CD56+ cells to feeder cells of about 1:1-100. For example, CD56+ cells can be co-cultured at a ratio of CD56+ cells to feeder cells of about 1:2, 1:5, 1:10, 1:30 or 1:100.

In some embodiments, IL-21 may be added at a concentration of 10-100 ng/mL during the first and/or second period. For example, IL-21 may be added at a concentration of 20-80 ng/mL, 30-70 ng/mL or 40-60 ng/mL during the first and/or second period. In some embodiments, IL-21 may be added one or more times during the first and/or second period.

In some embodiments, a method of propagating natural killer cells in culture comprises isolating CD56+ from a blood sample; co-culturing CD56+ cells with one or more feeder cells in the presence of IL-21; freezing the CD56+ cells; thawing the frozen CD56+ cells; and propagating the thawed CD56+ cells.

In some embodiments, CD56+ cells may be frozen at a temperature of less than -100 °C. In some embodiments CD56+ cells can be frozen at a temperature of -10 °C or less, -20 °C or less, -50 °C or less, -70 °C or less, -150 °C or less, -192 °C or less, or -200 °C or less. In some embodiments, frozen CD56+ cells may be stored for one or more days prior to thawing. In some embodiments, the frozen CD56+ cells are at least 2 days, at least 3 days, at least 7 days, at least 14 days, at least 30 days, at least 60 days, inclusive of any range between any two of the following values; or for 180 days or more. For example, frozen CD56+ cells can be stored for a period of more than one day and less than 10 years.

In some embodiments, CD56+ cells can be co-cultured for 13-16 days prior to freezing. For example, CD56+ cells can be co-cultured for 14 or 15 days prior to freezing. In some embodiments, co-culture or propagation (including re-proliferation) is 9-25 days, e.g., 10-24, 11-23, 13-22, 14-21, 14-18, 14-16 days. etc., may be during. These time frames can be applied to any of the proliferation and/or re-proliferation periods provided herein (including examples for other cells).

In some embodiments, the one or more feeder cells are selected from the group consisting of irradiated Jurkat cells, irradiated Epstein-Barr virus transformed lymphocyte continuous cell line (EBV-LCL) cells, K562 cells, and PBMCs. There may be more than one. CD56+ cells can be co-cultured at a ratio of CD56+ cells to feeder cells of about 1:1-100. For example, CD56+ cells can be co-cultured at a ratio of CD56+ cells to feeder cells of about 1:2, 1:5, 1:10, 1:30 or 1:100.

In some embodiments, IL-21 may be added at a concentration of 10-100 ng/mL. For example, IL-21 may be added at a concentration of 20-80 ng/mL, 30-70 ng/mL or 40-60 ng/mL. In some embodiments, IL-21 may be added one or more times.

In some embodiments, a method of increasing the cytotoxicity of natural killer cells comprises providing said natural killer cells; freezing the natural killer cells; thawing the frozen natural killer cells; and co-culturing the thawed natural killer cells with one or more feeder cells in the presence of IL-21.

In some embodiments, the method may further comprise storing the frozen natural killer cells at a temperature of less than -100 °C. In some embodiments, the frozen natural killer cells are stored at a temperature of -10 °C or lower, -20 °C or lower, -50 °C or lower, -70 °C or lower, -150 °C or lower, -192 °C or lower, or -200 °C or lower. can In some embodiments, frozen natural killer cells may be stored for one or more days prior to thawing. In some embodiments, the frozen natural killer cells are at least 2 days, at least 3 days, at least 7 days, at least 14 days, at least 30 days, at least 60 days, inclusive of any range between any two of the following values: , or for 180 days or more. In some embodiments, once thawed, cells are stored as long as there are viable cells.

In some embodiments, the one or more feeder cells are selected from the group consisting of irradiated Jurkat cells, irradiated Epstein-Barr virus transformed lymphocyte continuous cell line (EBV-LCL) cells, K562 cells, and PBMCs. There may be more than one. In some embodiments, thawed natural killer cells can be co-cultured at a ratio of natural killer cells to feeder cells of about 1:1-100.

In some embodiments, IL-21 may be added at a concentration of 10-100 ng/mL. For example, IL-21 may be added at a concentration of 20-80 ng/mL, 30-70 ng/mL or 40-60 ng/mL. In some embodiments, IL-21 may be added one or more times.

In some embodiments, a method of producing natural killer cells may comprise repeating the following steps: freezing; thawing step; and a second culturing step comprising co-culturing by adding feeder cells.

In some embodiments, one or more cycles of re-stimulation or re-proliferation may be applied, as shown in FIG. 14A (at bar time points above the data curve). In some embodiments, a first stimulation followed by cell culture followed by freezing (selective), followed by a second stimulation (re-stimulation), followed by a second cell culture step, followed by freezing (selective), followed by a third stimulation (second re-stimulation) -stimulation) followed by another incubation step. IL-21 can be used in each of the stimulation steps provided herein. The optional freezing step can be applied to all of the cells, or to some of the cells. In some embodiments, there are 2, 3, 4, 5, 6, 7, 8, 9, 10 or more stimulations (e.g., 1, 2, 3, 4, 5, 6, 7, 1, 2, 3, 4, 5, 6, 7, 8, 9 or more re-stimulations). After each re-stimulation, there may be another cell culture/proliferation step. In some embodiments, each cell culture or re-proliferation can proceed for 9-25 days. Each cell culture step may be followed by a freezing step. In some embodiments, the process may be a cycle consisting of: a) stimulation (or re-stimulation) followed by b) cell culture followed by c) freezing (optional) followed by d) thawing (optional), as desired. repeat. as much as you want. In some embodiments, the amount of IL-21 is between 10 and 100 ng/mL, such as 50 ng/mL. The stimulation/restimulation of Figure 14A shows the addition of IL-21 to the cells.

In some embodiments, any of the processes provided herein can include one or more freezing steps.

In some embodiments, any of the embodiments of NK cells provided herein may include native NK cells and genetically modified NK cells.

Cell therapy composition for cancer treatment

According to some embodiments, a cell therapy composition for the treatment of cancer may comprise peripheral blood derived CD56+ NK cells. The cells have undergone or will be the result of at least two stages of proliferation, with at least the first stage proceeding in the presence of IL-21.

As used herein, the term "peripheral blood-derived" means that cells are derived from "whole blood from peripheral blood" or "leukocytes isolated from peripheral blood using leukapheresis". can mean Peripheral blood-derived CD56+ NK cells can be used interchangeably with peripheral blood mononuclear cell (PBMC)-derived CD56+ NK cells.

In some embodiments, the cytokine is 18-180,000, 20-100,000, 50-50,000, 50-1,000, 50-900, 50-800, 50-700, 50-600, 50-550, 100-550, 150- Concentrations of 550, 200-550, 250-550, 300-550, 350-550, 400-550, 450-550 IU/mL may be used. When the cytokine is used in the above ranges, it is possible to inhibit apoptosis of NK cells included in the cancer treatment composition and increase the anti-cancer activity of NK cells.

In some embodiments, the composition can include IL-2 as an additional cytokine (eg, in addition to IL-21).

In some embodiments, CD56+ NK cells can be obtained as described elsewhere herein. For example, CD56+ NK cells can be obtained by co-culture with feeder cells (eg, irradiated Jurkat cells and irradiated EBV-LCL cells). In some embodiments, the ratio (purity) of CD56+ NK cells to total cells may be at least 85%, at least 90%, at least 95%, or at least 98%.

In some embodiments, the cancer can be, but is not limited to, blood cancer, stomach cancer, pancreatic cancer, cholangiocarcinoma, colon cancer, breast cancer, liver cancer, ovarian cancer, lung cancer, kidney cancer, prostate cancer, or neuroblastoma. In some embodiments, the process may be applied to allogeneic NK cell therapy, eg, in neurodegenerative diseases and acute infections.

In some embodiments, the composition may contain no T cells, or may contain only trace amounts of T cells. For example, the ratio of T cells to total cells in the composition may be less than 15%, less than 10%, less than 5%, less than 2%, less than 1% or less.

As used herein, the term "T cell (T cell)" refers to a lymphocyte derived from the thymus, which "memorizes" previously encountered antigens and provides information to B cells, thereby promoting antibody production and It plays an important role in the cellular immune system. Since these T cells can induce an immune response to allogeneic antigens by discriminating very small differences between different antigens, self-treatment is possible, but there may be limitations in using it for allogeneic treatment. Thus, a cell therapy composition free of T cells may be suitable for allograft.

As used herein, the term "cell therapeutic agent" refers to autologous, allogeneic and xenogeneic living cells in vitro to restore the function of cells and tissues or otherwise change the biological properties of the cells. It refers to a drug used for treatment, diagnosis and prevention through a series of actions, such as proliferation and screening. Cell therapies have been regulated as pharmaceuticals since 1993 in the United States and 2002 in Korea. These cell therapeutics can be broadly divided into two fields: first, stem cell therapeutics for tissue regeneration or restoration of organ functions, and second, immune responses such as suppression of immune response or enhancement of immune response in vivo. Regulating immune cell therapies.

The route of administration of the cell therapy compositions described herein can be any suitable route so long as the composition reaches the target tissue. Administration may be parenteral administration, for example, intraperitoneal administration, intravenous administration, intramuscular administration, subcutaneous administration, or intradermal administration, but is not limited thereto.

The cell therapy compositions described herein may be formulated in a suitable form with a pharmaceutically acceptable carrier suitable for cell therapy or commonly used. "Pharmaceutically acceptable" refers to a composition that is physiologically acceptable and does not generally cause gastrointestinal disorders, allergic reactions such as dizziness, or similar reactions when administered to the human body. Pharmaceutically acceptable carriers may include carriers for parenteral administration, such as, for example, water, suitable oils, saline, aqueous glucose and glycol, and may further contain stabilizers and preservatives. Suitable stabilizers include sodium bisulfite, sodium sulfite or antioxidants such as ascorbic acid, sucrose, albumin and the like. Suitable preservatives include DMSO, glycerol, ethylene glycol, sucrose, trehalose, dextrose, polyvinylpyrrolidone, and the like.

The cell therapy composition may also be administered by any device capable of delivering the cell therapy to the target cell.

A cell therapy composition may comprise a therapeutically effective amount of a cell therapeutic agent for the treatment of a disease. The term “therapeutically effective amount” refers to the amount of an active ingredient or cell therapy composition that elicits a biological or medical response in tissue systems, animals or humans under consideration by a researcher, veterinarian, physician or other clinician. is meant and includes an amount that induces alleviation of the symptoms of the diseases or disorders to be treated. It will be apparent to those skilled in the art that the cell therapeutic agent included in the cell therapy composition may be changed according to a desired effect. Therefore, the optimal content of the cell therapeutic can be easily determined by a person of ordinary skill in the art, and the type of disease, the severity of the disease, the content of other components included in the composition, the type of formulation, age, weight, general It can be adjusted according to various factors including health status, sex, patient's diet, administration time, administration route, secretion rate of the composition, treatment period, and concurrently used drugs. Considering all factors, it is important to include the amount that can achieve the maximum effect with the minimum amount without side effects. For example, the cell therapy composition may comprise 1 x 10 ⁶ to 5 x 10 ⁸ cells per kg body weight of the cell therapy.

In some embodiments, the NK cells of the cell therapy composition are 50% or more, 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 93% or more, 95% or more compared to a pre-frozen population thereof. % or greater, or 98% or greater cytotoxicity.

In some embodiments, the composition may comprise an effective amount of CD56+ cells derived from peripheral blood mononuclear cells (PBMCs) from a patient. CD56+ cells can be obtained by isolating peripheral blood mononuclear cells (PBMCs) from a blood sample; isolating CD56+ cells from PBMCs; co-culturing CD56+ cells with one or more feeder cells in the presence of one or more cytokines; freezing the CD56+ cells; thawing the frozen CD56+ cells; and co-culturing the thawed CD56+ cells with one or more feeder cells in the presence of one or more cytokines.

In some embodiments, an effective amount of CD56+ cells can be 1 x 10 ⁶ to 5 x 10 ⁸ cells per kg body weight.

In some embodiments, the cell composition comprises an effective amount of CD56+ cells derived from peripheral blood mononuclear cells (PBMCs) from a patient; IL-2; and IL-21.

In some embodiments, the composition comprises a first population of CD56+ cells derived from peripheral blood mononuclear cells (PBMCs); ice; IL-2 and IL-21. When thawed, CD56+ cells have at least 80% cytotoxicity of a second population of CD56+ cells, wherein the second population of CD56+ cells has not been frozen. In some embodiments, the cytotoxicity is at least 85, 90, 95, 96, 97, 98, or 99%.

In some embodiments, when comparing non-frozen proliferation (in the presence and absence of IL21, IL21+, or IL21-) to proliferation frozen but co-cultured with IL21+ in a first step, the mean cytotoxicity of the frozen proliferation is non- 97.7% of frozen (Table 3: Range 83%-131%). In some embodiments, when comparing non-frozen proliferation (in the presence and absence of IL21, IL21+ or IL21-) to proliferation that is frozen but not co-cultured with IL21+ in a first step (IL21--, IL21-+), The average cytotoxicity of frozen proliferations can be at least 81.4% of non-frozen proliferations (Table 4 range 61%-83%). When IL21 was added to both stages of frozen proliferation (IL21++), the mean cytotoxicity was 114% of non-frozen proliferation (Table 3 values 98%-131%). Thus, in some embodiments, IL21+/+ (72%) versus IL21 −/+ (45%) is less cytotoxic in the first phase when proliferating with IL21 and in the second phase than without Il21 in the first phase. indicates that it is 60% higher. Thus, in some embodiments, the presence of IL21 in the first proliferation allows for a second proliferation after freezing that is 60% higher. For IL21+/- (62%) versus IL21 −/- (46.1%), when it first proliferates with IL21 in the first phase but not in the second phase, cytotoxicity is This means that it can be 35% or more higher than without.

In some embodiments, the cytotoxicity is a comparison of non-frozen proliferation (with or without IL21) versus frozen but co-cultured with IL21+ in a first step, wherein the mean cytotoxicity of the frozen proliferation is non- It is 97.7% of frozen growths. In some embodiments, the cytotoxicity is a comparison of non-frozen proliferation (in the presence or absence of IL21) versus proliferation that has been frozen but not co-cultured with IL21+ in a first stage, wherein the mean cytotoxicity of the frozen proliferation is the ratio - 81.4% of frozen propagation. In some embodiments, IL21 is added both before and after freezing, and the mean cytotoxicity is 114% of non-frozen proliferation.

In some embodiments, the superior properties of initial IL21 treatment during co-proliferation to allow for subsequent re-proliferation may be consistent with the results in Tables 1-4 below:

Cytotoxic k562 % Lysis 10:1 3:1 1:1 0.5:1 IL21+ 95.8 98.8 74.6 53.3 IL21- 94.2 81.4 55.6 37.7 IL21+/+ 98.1 84.9 72.8 42.9 IL21+/- 99.6 83.2 62.2 33.6 IL21-/+ 89.1 81 45.3 31.2 IL21-/- 91.6 77.3 46.1 30.8

Comparison with the presence of IL21 in the first step 10:1 3:1 1:1 0.5:1 AVG 2-step frozen / 2-step frozen IL21++ / IL21-- 107% 110% 158% 139% 129% 2-step frozen / 2-step frozen IL21++ / IL21+- 98% 102% 117% 128% 111% 2-step frozen / 2-step frozen IL21++ / IL21-+ 110% 105% 161% 138% 128% 2-step frozen / 2-step frozen IL21+- / IL21-- 109% 108% 135% 109% 115%

Comparison with the presence of IL21 in the first step 10:1 3:1 1:1 0.5:1 AVG 2-step frozen / 1-step Unfrozen IL21++ / IL21+ 102% 86% 98% 80% 92% 2-step frozen / 1-step Unfrozen IL21++ / IL21- 104% 104% 131% 114% 113% 2-step frozen / 1-step Unfrozen IL21+- / IL21+ 104% 84% 83% 63% 84% 2-step frozen / 1-step Unfrozen IL21+- / IL21- 106% 102% 112% 89% 102% Total Average: 97.70%

Comparison with the absence of IL21 in the first step 10:1 3:1 1:1 0.5:1 AVG 2-step frozen / 1-step Unfrozen IL21--/IL21- 97% 95% 83% 82% 89% 2-step frozen / 1-step Unfrozen IL21--/IL21+ 96% 78% 62% 58% 73% 2-step frozen / 1-step Unfrozen IL21-+/IL21- 95% 100% 81% 83% 90% 2-step frozen / 1-step Unfrozen IL21-+/IL21+ 93% 82% 61% 59% 74% Total Average: 81.40%

In any embodiment for culturing, proliferating (including re-proliferation) NK cells, the number of peripheral blood mononuclear cells in the culture at the start of the co-culture (cultivation, proliferation (including re-proliferation)) is between 1x10 ⁴ and 1x10 ¹⁵ range of cells. In some embodiments, the number of peripheral blood mononuclear cells in the culture medium at the start of the co-culture is 1x10 ⁴ to 5x10 ⁴ cells, 5x10 ⁴ to 1x10 ⁵ cells, 1x10 ⁵ to 5x10 ⁵ cells, 5x10 ⁵ to 1x10 ⁶ cells, 1x10 ⁶ to 1x10 ⁷ cells, 1x10 ⁷ to 1x10 ⁸ cells, 1x10 ⁸ to 1x10 ⁹ cells, 1x10 ⁹ to 1x10 ¹⁰ cells, 1x10 ¹¹ to 1x10 ¹² cells, 1x10 ¹² to 1x10 ¹³ cells, 1x10 ¹³ to 1x10 ¹⁴ cells, or 1x10 ¹⁴ to It is in the range of 1x10 ¹⁵ cells. In some embodiments, the number of peripheral blood mononuclear cells in the culture at the beginning of the first or initial proliferation is 1x10 ⁴ to 5x10 ⁴ cells, 5x10 ⁴ to 1x10 ⁵ cells, 1x10 ⁵ to 5x10 ⁵ cells, 5x10 ⁵ to 1x10 ⁶ cells, 1x10 ⁶ to 1x10 ⁷ cells, 1x10 ⁷ to 1x10 ⁸ cells, 1x10 ⁸ to 1x10 ⁹ cells, 1x10 ⁹ to 1x10 ¹⁰ cells, 1x10 ¹¹ to 1x10 ¹² cells, 1x10 ¹² to 1x10 ¹³ cells, 1x10 ¹³ to 1x10 ¹⁴ cells, or It ranges from 1x10 ¹⁴ to 1x10 ¹⁵ cells. In some embodiments, the number of peripheral blood mononuclear cells in the culture at the start of re-proliferation is 1x10 ⁴ to 5x10 ⁴ cells, 5x10 ⁴ to 1x10 ⁵ cells, 1x10 ⁵ to 5x10 ⁵ cells, 5x10 ⁵ to 1x10 ⁶ cells, 1x10 ⁶ to 1x10 6 cells. 1x10 ⁷ cells, 1x10 ⁷ to 1x10 ⁸ cells, 1x10 ⁸ to 1x10 ⁹ cells, 1x10 ⁹ to 1x10 ¹⁰ cells, 1x10 ¹¹ to 1x10 ¹² cells, 1x10 ¹² to 1x10 ¹³ cells, 1x10 ¹³ to 1x10 ¹⁴ cells, or 1x10 ¹⁴ to It is in the range of 1x10 ¹⁵ cells. The present methods can provide for greater proliferation of NK cells than existing approaches. Thus, in any of the above embodiments, the number of peripheral blood mononuclear cells in the culture at the start of the co-culture (culture, proliferation (including re-proliferation)) is such that the number of peripheral blood mononuclear cells in the culture medium causes NK cells to proliferate (eg, IL-21 and/or or proliferate without cytokines such as IL-2) to provide a similar number of NK cells (eg, for therapeutic use and/or cryopreservation), which is difficult to use in existing approaches. Likewise, in some embodiments the methods of the present disclosure provide NK cells suitable for therapeutic use, eg, immunotherapy. In some embodiments, the methods of the present disclosure provide for cryopreservation of NK cells that can be subsequently thawed and effectively propagated for therapeutic use. Thus, in any embodiment for culturing or propagating (including re-proliferation) NK cells, the NK cells are propagated to produce one population of proliferated NK cells for therapeutic use and another population for cryopreservation. . In some embodiments, cryopreserved NK cells are later thawed and re-proliferated for therapeutic use and/or further cryopreservation.

In any embodiment for culturing or proliferating (including re-proliferating) NK cells, the number of NK cells at the end of proliferation or re-proliferation is greater than the number of NK cells effective for therapeutic use. In some embodiments, excess NK cells are cryopreserved for future use, eg, for future thawing, proliferation, and administration to a patient in need thereof. In some embodiments, the NK cells are at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, or proliferate or re-proliferate to a greater extent than the number of NK cells to be used or to be used in treatment, eg, immunotherapy, above, or as a percentage in the range between any two of the previous values.

In any embodiment for culturing or propagating (including re-proliferation) NK cells, the method may comprise repeating a freeze-thaw-proliferation cycle. In some embodiments, the method comprises repeating the freeze-thaw-growth cycle 1, 2, 3, 4, 5, 6, 7, 8 or more times.

Also provided herein is a composition of cryopreserved NK cells, wherein the NK cells retain biological activity, eg, cytotoxicity, even after thawing. In some embodiments, cryopreserved NK cells retain their biological activity, eg, cytotoxicity, after thawing and re-proliferation. In some embodiments, the composition of cryopreserved NK cells is prepared by any of the methods for culturing or propagating (including re-proliferating) NK cells, as disclosed herein. In some embodiments, the composition comprises a population of immune cells that are at least 80%, 85%, 90%, 95%, 97% or more NK cells. The composition may contain a suitable cryopreservation medium. In some embodiments, the composition comprises dimethylsulfoxide (DMSO) and serum (eg, FBS, human serum). In some embodiments, the composition comprises 1-15%, 2-15%, 5-15%, 5-10%, or about 10% DMSO. In some embodiments, the composition comprises or consists of a population of cryopreserved immune cells comprising at least 90% NK cells, 10% DMSO and 90% FBS. In some embodiments, the NK cells are derived from PBMCs obtained from a subject. In some embodiments, the composition comprises or consists of a population of cryopreserved immune cells comprising at least 90% NK cells, 5-10% DMSO and 90-95% FBS. In some embodiments, the NK cells are derived from PBMCs obtained from a subject. In some embodiments, it further comprises a CryoStor solution.

How to prevent or treat cancer

In some embodiments, methods of preventing or treating cancer are provided. The method comprises administering to a subject an anti-cancer cell therapy composition comprising peripheral blood-derived CD56+ natural killer cells and cytokines. In some embodiments, the cell is the result of a dual-proliferative process, at least the first of which occurs in the presence of IL-21.

The term “subject” refers to a mammal, preferably a human, that is a subject for treatment, observation or testing. The subject may be a patient with blood cancer, stomach cancer, pancreatic cancer, cholangiocarcinoma, colon cancer, breast cancer, liver cancer, ovarian cancer, lung cancer, kidney cancer, prostate cancer, or neuroblastoma, but is not limited thereto.

In some embodiments, for adults, the cell therapy composition may be administered from once to several times a day. The cell therapy composition may be administered daily or at 2-180 day intervals. The cell therapeutic agent included in the composition may comprise 1 x 10 ⁶ to 1 x 10 ¹¹ peripheral blood-derived CD56+ natural killer cells, eg, about 1 x 10 ⁶ to 1 x 10 ⁸ NK cells per kg body weight. . In some embodiments, the peripheral blood-derived CD56+ natural killer cells in the cell therapy composition are at least about 90% pure. In some embodiments, the cytokine is IL-2 at a concentration ranging from about 50 - 50,000 IU/ml.

In some embodiments, cell therapy compositions may be formulated in a suitable form with a pharmaceutically acceptable carrier suitable for or commonly used in cell therapy. “Pharmaceutically acceptable” refers to a composition that is physiologically acceptable and does not generally cause gastrointestinal disorders, allergic reactions such as dizziness, or similar reactions when administered to the human body. Pharmaceutically acceptable carriers include, for example, carriers for parenteral administration such as water, suitable oils, saline, aqueous glucose, glycols, basic compounds such as Hartman's solution, or alternatives such as common saline solution, plasma electrolyte A and the like. and may further include stabilizers and preservatives. Suitable stabilizers include sodium bisulfite, sodium sulfite, or antioxidants such as ascorbic acid, sucrose, albumin, human serum albumin, and the like. Suitable preservatives include DMSO, glycerol, ethylene glycol, sucrose, trehalose, dextrose, polyvinylpyrrolidone, and the like.

In some embodiments, the cell therapy composition can be administered by any suitable method, such as administration via rectal, intravenous, intraarterial, intraperitoneal, intramuscular, intrasternal, transdermal, topical, intraocular, or intradermal routes. In some embodiments, the NK cells comprised in the composition may be allogeneic, i.e., obtained from a person other than the subject of treatment. In some embodiments, the human may be a normal person or a cancer patient. In some embodiments, the NK cells included in the composition may be autologous, ie, obtained from the subject being treated.

In some embodiments, the NK cells disclosed herein and a cell therapy composition comprising the NK cells disclosed herein may be used to treat a disease or condition other than cancer. NK cells have been reported to play an important role in the regulation of the immune system, for example by modulating T-cells, and thus a cell therapy composition comprising NK cells can be administered to treat conditions associated with the immune system. . For example, the cell therapy composition may be used for neurodegenerative disorders (eg, Alzheimer's disease and Parkinson's disease) or autoimmune diseases (eg rheumatoid arthritis, multiple sclerosis, psoriasis, spondyloarthropathies, SLE, Sjogren's syndrome, systemic sclerosis).

In some embodiments, a method of treating a subject comprises collecting CD56+ cells from the subject; co-culturing CD56+ cells with one or more feeder cells in the presence of IL-21; freezing the co-cultured CD56+ cells for at least one day; thawing the frozen CD56+ cells; propagating thawed CD56+ cells; and administering the proliferated CD56+ cells to the subject, wherein the cytotoxicity of the cells from the second proliferation is at least 80% (eg, 80, 85, 90 ) of the cytotoxicity of the co-cultured CD56+ prior to freezing. , 95, or 97% or more).

In some embodiments, the method may further comprise storing the frozen CD56+ cells at a temperature of less than -100 °C. In some embodiments, the frozen CD56+ cells are stored at a temperature of -10 °C or less, -20 °C or less, -50 °C or less, -70 °C or less, -150 °C or less, -192 °C or less, or -200 °C or less. can In some embodiments, frozen CD56+ cells may be stored for one or more days prior to thawing. In some embodiments, the frozen CD56+ cells are at least 2 days, at least 3 days, at least 7 days, at least 14 days, at least 30 days, at least 60 days, inclusive of any range between any two of the following values; or for 180 days or more.

In some embodiments, CD56+ cells can be co-cultured for 13-16 days prior to freezing. For example, CD56+ cells can be co-cultured for 14 or 15 days prior to freezing. In some embodiments, co-culturing or propagation (including re-proliferation) is 9-25 days, e.g., 10-24, 11-23, 13-22, 14-21, 14-18, 14-16 days. , and so on. These time frames can be applied to any of the proliferation and/or re-proliferation periods provided herein (including examples for other cells).

In some embodiments, propagating the thawed CD56+ cells comprises co-culturing the thawed CD56+ with one or more irradiated feeder cells in the presence of IL-21. The one or more feeder cells are one or more selected from the group consisting of irradiated Jurkat cells, irradiated Epstein-Barr virus transformed lymphocyte continuous cell line (EBV-LCL) cells, K562 cells and PBMCs. In some embodiments, CD56+ cells may be co-cultured at a ratio of CD56+ cells to feeder cells of about 1:1-100. For example, CD56+ cells can be co-cultured at a ratio of CD56+ cells to feeder cells of about 1:2, 1:5, 1:10, 1:30 or 1:100.

In some embodiments, IL-21 may be added at a concentration of 10-100 ng/mL during the first and/or second period. For example, IL-21 may be added at a concentration of 20-80 ng/mL, 30-70 ng/mL or 40-60 ng/mL during the first and/or second period. In some embodiments, IL-21 may be added one or more times during the first and/or second period.

In some embodiments, the IL-21 is human IL-21 on human NK cells.

Some embodiments provided herein include the following features and advantages:

(a) A method of producing natural killer cells.

(b) Even after cryopreservation, natural killer cells can be produced in a sufficient amount for clinical use, thereby increasing the effectiveness of cancer prevention and treatment, in particular, allopathic therapy using natural killer cells.

(c) the cytotoxicity of the resulting cells proliferated twice (at least the first with IL-21, and optionally in the second propagation) was compared to cells propagated without IL-21 (first, much larger twice). much better than In some embodiments, the cells are in 1-50 mL cryo-vials, or in 10-100 mL cryo-bags.

Methods have been developed and provided herein to help NK cells undergo and recover from cryopreservation. It has been found that CD56+ cells can be successfully propagated after freezing and thawing when CD56+ cells are co-cultured with feeder cells in the presence of IL-21 initially (during initial proliferation). By using IL-21, high-purity CD56+ NK cells can be generated, and surprisingly, they retain a particularly high amount of cytotoxicity. Moreover, after the NK cells have been thawed, they can be further expanded (without IL-21, or even more advantageously, in the presence of additional IL-21). Thus, IL-21 in the pre-freezing process (eg first propagation) may allow good re-proliferation later. In addition, the resulting product, even after being propagated twice and frozen once, retains a surprisingly high amount of cytotoxicity, which is more pronounced when IL-21 is used for the first as well as the second growth. is strengthened In some embodiments, this freezing and re-growth process may be repeated multiple times (each time, optionally with another step of IL-21 during re-proliferation).

Various embodiments are shown in the accompanying drawings for purposes of illustration, and should not be construed as limiting the scope of the embodiments. Moreover, various features of the different disclosed embodiments may be combined to form further embodiments, which are part of the present disclosure.
1 shows the design of a re-proliferation experiment and embodiments for cell proliferation after IL21 treatment (pre-frozen and optionally post-frozen).
2A and 2B show a comparison of the population doubling level (PDL) between cell proliferation of cells with and without IL21 ( FIG. 2A ) Donor 1 , ( FIG. 2B ) Donor 2.
Figures 3a and 3b show a comparison of proliferation folds between cell proliferation in cells with and without IL21 (Figure 3a) Donor 1, (Figure 3b) Donor 2.
4A and 4B show a comparison of population doubling levels (PDL) between cell re-stimulation methods of cells with and without IL21 ( FIG. 4A ) Donor 1 , ( FIG. 4B ) Donor 2.
5a and 5b show a comparison of proliferation fold between cell re-stimulation methods of cells with and without IL21 ( FIG. 5a ) donor 1 , ( FIG. 5b ) donor 2.
6 shows the cytotoxic activity of NK cells against (IL-21+) K562 cells proliferated with IL-21.
7 shows the cytotoxic activity of NK cells against (IL-21-) K562 cells proliferated without IL-21.
Figure 8 shows the cytotoxic activity of NK cells against (IL-21+/+) K562 cells proliferated with IL-21 and re-stimulated with IL-21.
Figure 9 shows the cytotoxic activity of NK cells against (IL-21+/-) K562 cells proliferated with IL-21 and re-stimulated without IL-21.
Figure 10 shows the cytotoxic activity of NK cells against (IL-21-/+) K562 cells proliferated without IL-21 and re-stimulated with IL-21.
11 shows the cytotoxic activity of NK cells against (IL-21-/-) K562 cells proliferated without IL-21 and re-stimulated without IL-21.
` FIG. 12A shows a phenotypic comparison of activating receptors of NK cells.
12B shows a comparison of phenotypic inhibition of NK cells and chemokine receptors.
13A shows NK cells proliferated with IL-21 and re-proliferated with IL-21 (IL-21+/+) and without IL-21 and re-proliferated without IL-21 (IL-21- //) shows a graph of the population doubling level (PDL) of NK cells.
13B shows NK cells proliferated with IL-21 and re-proliferated with IL-21 (IL-21+/+) and proliferated with IL-21 and re-proliferated without IL-21 (IL-21). +/-) A graph of the population doubling level (PDL) of NK cells is shown.
14A shows a graph of the population doubling level (PDL) of NK cells proliferated with IL-21 (first stimulation) and re-proliferated two or more times (second and third stimulation).
FIG. 14B shows a graph of proliferation folds corresponding to the results in FIG. 14A .

The following examples are provided to illustrate certain features and/or embodiments. These examples should not be construed as limiting the disclosure to the specific features or embodiments described.

A comparative test for the method for producing high-purity NK cells was performed using LCL+KL-1 type feeder cells with or without IL-21 treatment. The other feeder cells discussed in this example are provided as prophetic examples.

The effect of IL-21 will be verified in two production methods: 1) original , incubating isolated CD56+ cells with other types of feeder cells under treatment or non-treatment with IL-21 for 14-17 days method (but no subsequent re-proliferation). 2) A re-stimulation method , culturing the cryopreserved proliferated NK cells in the original method with other types of feeder cells under treatment or non-treatment with IL-21. Different types of feeder cells and preparation methods are shown in Table 5 and FIG. 1 , which is generally understood to be representative of feeder cells for NK cell proliferation.

Feeder cell types and processes for NK cell culture Items Innoculation Ratio original method Re-stimulation method (NK cell: Feeder cell) Feeder cell type LCL+KL-1 cells 1:30:30 CD56 positive cells isolated from fresh or cryopreserved PBMCs NK cells cryopreserved at 14 or 15 days K562 cells 1:10 PBMCs 1:10 Culture Condition + IL-21 Co-culture with irradiated feeder cells and IL-21 for 0-6 days - IL-21 Co-culture with radiation feeder cells without IL-21

It is understood that the results provided by this example are generally representative of feeder cells across various ratios, storage times and additional components (eg additional cytokines) as long as Il-21 is present.

Example 1: Separation of starting materials and original method

Peripheral blood mononuclear cells (PBMCs) were obtained from human blood. Isolated PBMCs were used for CD56+ cell selection. PBMCs were isolated by density gradient centrifugation using 1.077 g/mL Ficoll, washed several times with PBS, and resuspended in AutoMACS Rinsing Solution (Miltenyi Biotec, Germany) containing CD56 microbead reagent. CD56+ cells were selected using a magnetically activated cell sorting (MACS) system according to the manufacturer's instructions (Miltenyi Biotec, Germany). CD56+ selected cells were resuspended in initial NK cell culture medium with or without 50 ng/mL IL-21. Floating cells were inoculated into culture flasks after addition of 100 Gy irradiated feeder cells, LCL+KL-1, K562 or PBMCs, and then cultured at 37° C. 5% CO ₂ for 6 or 7 days. Specific conditions of cell culture for each trophic type are shown in the table below.

Culture conditions for NK cell proliferation Feeder cell type Feeder cell ratio Culture media and supplements (CD56+ cells: Feeder cells) LCL+KL-1 1:30: 30 at D0 and D3 RPMI media, 10% FBS, 500 IU/mL IL-2, 20 ug/mL gentamicin Autologous PBMCs ¹⁾ 1:5 at D0 and D7 CellGro SCGM medium, 1% autologous plasma or 10% FBS, 10 ng/ml anti-CD3 monoclonal antibody OKT3, 500 IU/mL IL-2, 20 ug/mL gentamicin K562 1:10 CellGro SCGM medium, 10 % FBS, 500 IU/mL IL-2, 20 ug/mL gentamicin

See: Immune Network. 2018 Aug;18(4):e31 On day 6 or 7 of culture, cells were collected from culture flasks by centrifugation and cell number was assessed. Cells were resuspended in NK cell culture medium, inoculated into culture bags, and then incubated at 37° C. in 5% CO ₂ for up to 17 or 18 days. Every 3 or 4 days, cells were sub-cultured with fresh medium.

Example 2: Cryopreservation of D14 cells

On day 14 or 15 of culture, cells were harvested from culture bags by centrifugation and cell number was assessed. Cells were resuspended in medium containing 90% FBS, 10% DMSO with or without 500 IU/mL IL-2, placed in vials at a concentration of 5.0 - 10.0 x 10 ⁶ cells/mL, and then cells were - They were cryopreserved for at least 1 week, 1, 3, 6, 12, 24 months in a liquid nitrogen tank at 196 °C.

Example 3: Cell Culture of Frozen Cell Thaw and Re-stimulation Method

The cryopreserved cells of the original method were thawed in a 37°C water bath and resuspended in initial NK cell culture medium containing supplements for each feeder cell condition with or without 50 ng/mL IL-21. Floating cells were seeded in culture flasks after addition of 100 Gy irradiated feeder cells in LCL+KL-1, K562 or autologous PBMCs, and then incubated at 37° C. in 5% CO ₂ for 6 or 7 days. This process is called re-stimulation. On day 6 or 7 of culture, cells were collected from culture flasks by centrifugation and cell number was assessed. Cells were resuspended in NK cell culture medium, inoculated into culture bags, and then incubated at 37° C. in 5% CO ₂ for up to 17 or 18 days. Every 3 or 4 days, cells were sub-cultured with fresh medium. The total cell culture period took 31-33 days from the original method of D0 through the re-stimulation process to the final harvest.

Example 4: Population Doubling Levels and Cell Growth

NK cell culture according to 6 different conditions was performed using CD56+ cells based on the experimental design as exemplified in FIG. 1 and Table 7. This represents the results of Experimental Design Type 1 as NK cells co-cultured with irradiated LCL and KL-1 as feeder cells. The specific conditions for the process are as indicated in Table 7.

culture conditions ID First step expansion Re-Expansion After Cryopreservation of Expanded NK Cells IL21+. (+) IL-21 N/A Type 1. IL21+/+ (+) IL-21 (+) IL-21 Type 1. IL21+/- (+) IL-21 (-) IL-21 IL21-. (-) IL-21 N/A Type 1. IL21-/+ (-) IL-21 (+) IL-21 Type 1. IL21-/- (-) IL-21 (-) IL-21

The cell proliferation rate of NK cells was evaluated by the proliferation fold relative to the number of seeded cells and the population doubling level (PDL) of each passage-culture day, calculated as 3.32 (log N - log No), where N is the number of cells in each passage. Number of cells at the end and No is the number of cells initially plated. The number of NK cells in all culture steps was assessed by staining the cells with trypan blue.

Production batches from two different donors were compared for PDL and fold fold. As shown in Tables 8, 9, and Figures 2A-3B, the PDL and proliferation folds of NK cells from one donor by the original method using IL-21 showed higher growth rates than those in the absence of IL-21. However, batches from different donors showed similar growth rates in both conditions.

PDL in the original method Donor ID D0 D6 D10 D14 D17 Donor 1 IL21+. 0.00 4.98 9.64 11.46 12.15 IL21-. 0.00 3.32 6.17 8.40 8.62 Donor 2 IL21+. 0.00 5.80 9.40 11.10 11.94 IL21-. 0.00 5.80 9.49 11.10 11.48

Multiplication of original method Donor ID D0 D6 D10 D14 D17 Donor 1 IL21+. One 32 800 2,833 4,560 IL21-. One 10 72 340 394 Donor 2 IL21+. One 56 680 2,200 3,960 IL21-. One 56 720 2,200 2,860

Cells cryopreserved at D14 in the original method were re-stimulated with feeder cells with or without IL-21 treatment and cultured for up to 17 or 18 days. The total cell culture period took 31-33 days from the original method of D0 through the re-stimulation process to the final harvest (see Fig. 1).

Different production batches from both donors were compared for PDL and proliferation folds. As shown in Tables 10, 11 and Figures 4A-5B, the fold proliferation of PDL and NK cells of the re-stimulation method with IL-21 gave higher growth rates than those without the IL-21 condition. Therefore, IL-21 is very useful, especially in the first stage of proliferation, to allow for more effective subsequent stages of propagation.

NK cells from donor 2 showed no difference in growth rates with and without IL-21 in the original method, but showed a higher growth rate in the IL-21 condition by the re-stimulation method than in the absence of IL-21 at day 31. appear.

PDL of re-stimulation method Donor ID D0 D6 D10 D14 D20 D24 D28 D31 Donor Type 1. 0 4.98 9.64 11.46 16.94 19.42 21.67 22.4 One IL21+/+ Type 1. 0 4.98 9.64 11.46 16.72 19.95 21.89 22.82 IL21+/- Type 1. 0 3.32 6.17 8.4 12.35 15.15 17.25 18.25 IL21-/+ Type 1. 0 3.32 6.17 8.4 11.02 14.25 16.63 18.12 IL21-/- Donor Type 1. 0 5.8 9.4 11.1 16.89 20.47 22.57 23.57 2 IL21+/+ Type 1. 0 5.8 9.4 11.1 16.94 20.53 22.48 23.33 IL21+/- Type 1. 0 5.8 9.49 11.1 16.5 19.94 21.53 22.11 IL21-/+ Type 1. 0 5.8 9.49 11.1 16.5 18.79 20.44 21.16 IL21-/-

Proliferation multiples of the re-stimulation method Donor ID D0 D6 D10 D14 D20 D24 D28 D31 Donor Type 1. One 32 800 2,833 126,754 708,333 3,355,263 5,592,105 One IL21+/+ Type 1. One 32 800 2,833 108,974 1,024,359 3,923,077 7,453,846 IL21+/- Type 1. One 10 72 340 5,231 36,615 156,923 313,846 IL21-/+ Type 1. One 10 72 340 2,092 19,615 102,000 287,692 IL21-/- Donor Type 1. One 56 680 2,200 122,222 1,466,667 6,285,714 12,571,429 2 IL21+/+ Type 1. One 56 680 2,200 126,923 1,523,077 5,923,077 10,661,538 IL21+/- Type 1. One 56 720 2,200 93,077 1,015,385 3,046,154 4,569,231 IL21-/+ Type 1. One 56 720 2,200 93,077 456,923 1,438,462 2,369,231 IL21-/-

Example 5: Purity of NK cells

NK cells are known to express CD56 and lack CD3. To investigate the purity of NK cells before and after proliferation by the original and re-stimulation methods, flow cytometry was applied. NK cells were stained with fluorochrome-labeled antibodies of anti-CD56-FITC and anti-CD3-PE, and then analyzed using flow cytometry.

As NK cell culture proceeded with the original and re-stimulation method in both conditions (with or without IL-21 treatment), the proportion of NK cells (CD56+CD3-) in the NK cells proliferated from two different donors was 31 It increased rapidly to over 99% on day one (Table 11). Cell surface markers of other cell types, such as CD3+, CD20+ and CD14+, appeared in very low populations at the final culture stage (Table 12).

NK cell marker expression (CD3-CD56+) pattern on culture-proliferated NK cells Donor ID Before Frozen After Thing D0 D10 D14 D17 D24 D28 D31 Donor 1 Type 1. IL21+/+ 75.23 98.31 99.24 99.27 99.35 99.65 99.60 Type 1. IL21+/- - - - - 98.67 99.24 99.70 Type 1. IL21-/+ 75.23 94.30 96.29 95.63 90.93 96.38 98.09 Type 1. IL21-/- - - - - 94.42 92.59 97.28 Donor 2 Type 1. IL21+/+ 82.83 98.60 98.20 98.83 98.68 99.36 99.86 Type 1. IL21+/- - - - - 98.16 99.69 99.84 Type 1. IL21-/+ 82.83 99.03 98.64 99.39 96.84 99.47 99.81 Type 1. IL21-/- - - - - 97.43 99.21 99.63

Other cell marker expression (CD3, CD20, CD14) patterns on culture-proliferated NK cells Donor ID Surface Marker Before Frozen After Thing D0 D10 D14 D17 D24 D28 D31 Donor 1 Type 1. CD3 24.1 0.38 0.2 0.48 0.28 0.06 0.22 IL21+/+ CD20 0.43 0.04 0.68 0.45 0.57 0.12 0.05 CD14 0.28 0.3 1.72 0.23 0.12 0.12 0.19 Type 1. CD3 - - - - 0.13 0.11 0.08 IL21+/- CD20 - - - - 0.16 0.14 0.05 CD14 - - - - 0.03 0.27 0.23 Type 1. CD3 24.1 2.03 2.21 3.38 0.85 0.26 0.61 IL21-/+ CD20 0.43 0.54 1.11 0.38 0.45 0.59 0.14 CD14 0.28 1.85 1.75 0.61 0.28 0.17 0.44 Type 1. CD3 - - - - 0.95 1.13 1.4 IL21-/- CD20 - - - - 0.88 0.51 0.21 CD14 - - - - 0.66 0.13 0.38 Donor 2 Type 1. CD3 16.96 1.13 1.4 1.08 0.86 0.41 0.1 IL21+/+ CD20 1.35 0.34 0.2 0.02 0.49 0.45 0.04 CD14 0.47 0.25 0.2 0.04 0.53 0.27 0.05 Type 1. CD3 - - - - 1.08 0.14 0.1 IL21+/- CD20 - - - - 0.85 0.12 0.03 CD14 - - - - 0.6 0 0.08 Type 1. CD3 16.96 0.59 1.08 0.55 0.87 0.11 0.07 IL21-/+ CD20 1.35 0.15 0.06 0.12 0.8 0.13 0.06 CD14 0.47 0.22 0.1 0.13 0.57 0.12 0.05 Type 1. CD3 - - - - 1.3 0.62 0.35 IL21-/- CD20 - - - - 1.17 0.54 0.13 CD14 - - - - 1.05 0.64 0.31

Example 6 Cytotoxic function of NK cells

Cytotoxicity of NK cells against tumor target cell lines was assessed by a fluorometric cytotoxicity assay. NK cells were co-cultured with K-562 cells stained with Calcein AM at E:T ratios of 10:1, 3:1, 1:1, and 0.5:1 under light protection for 4 hours. RPMI1640 containing 10% FBS or 2% Triton X100 was added to target cells to provide spontaneous and maximal release. For calcein release assays, the supernatant was harvested after incubation of target cells and NK cells, and fluorescence was assessed using a SpectraMax M2 microplate reader (Molecular devices, San Jose, CA). The percent specific dissolution was calculated using the formula [(test release-spontaneous release)/(maximum release-spontaneous release)] x 100.

The cytotoxicity of NK cells cultured by the original and re-stimulation method was tested using the standard K-562 cell line, an NK sensitive target. Cells proliferated in the original method with and without IL-21 showed strong cytotoxic activity against K-562 even at low E:T ratios (1:1 and 0.5:1) ( FIGS. 6-7 ). However, the cytotoxic activity of NK cells by the re-stimulation method was different depending on the presence or absence of IL-21 treatment. NK cells treated with IL-21 for both the original method and the re-stimulation method showed stronger cytotoxic activity than the other conditions ( FIGS. 8-11 ).

However, in both the original and re-stimulation conditions, NK cells in the non-IL-21 condition showed decreased cytotoxic activity at an E:T ratio of 0.05:1 to 3:1. In addition, it showed a lower level of cytotoxicity in the E:T ratio of 10:1 than in the IL-21 treatment condition.

NK cells proliferated with IL-21 treatment showed very strong cytotoxicity against the K-562 cell line, and similar cytotoxicity to both NK cells prepared by the original and re-stimulation method (Figs. 6-11). ). 6 shows the cytotoxic activity of NK cells against IL-21 co-proliferated (IL-21+) K562 cells. 7 shows the cytotoxic activity of NK cells against (IL-21-) K562 cells proliferated without IL-21. 8 shows the cytotoxic activity of NK cells against K562 cells proliferated with IL-21 and re-stimulated with IL-21 (IL-21+/+). 9 shows the cytotoxic activity of NK cells against K562 cells proliferated with IL-21 and re-stimulated without IL-21 (IL-21+/-). Figure 10 shows the cytotoxic activity of NK cells against K562 cells proliferated without IL-21 and re-stimulated with IL-21 (IL-21-/+). 11 shows the cytotoxic activity of NK cells against K562 cells proliferated without IL-21 and re-stimulated without IL-21 (IL-21-/-). Figure 12A shows a phenotypic comparison of the activating receptors of NK cells. 12B shows a phenotypic comparison of inhibition of NK cells and chemokine receptors.

surface marker expression

NK cell function is finely regulated by a balance between surface-expressed activating and inhibitory receptors. Activation of [CD16, NKp30, NKp46, NKp44, NKG2D, 2B4 (CD244), NKG2C, CRACC] or inhibitory NK receptors [NKG2A, KIR: CD158a (KIR2DL1), CD158b (KIR2DL2/L3), CD158e (KIR3DL1)] To phenotypically characterize expression levels, chemokine receptors (CXCR3, CXCR4), or adhesion molecules (CD62L) were pre-proliferated (Day) by the original (Old) process and re-stimulation process with IL-21 treatment for 17-18 days. 0; DO) and post-proliferation gated CD56+ NK cells. Surface receptor expression levels were calculated as the percentage of receptor-positive subsets of NK cells in the samples. At each stage of the original and re-stimulation process, NK cells were stained with fluorochrome-labeled antibodies of each marker, and then analyzed using a flow cytometer.

Surface receptor expression levels were analyzed in cells at the beginning and end stages (D0, D17 and D32) incubated with the original and re-stimulation procedures with IL-21 treatment in type 1 experiments on 4 different donors. Surface receptor expression levels were calculated as the percentage of receptor-positive subsets of NK cells in the samples.

Among the activating receptors, the expression levels of CD16, NKp30, NKp46, NKp44, NKG2D and CRACC were increased during proliferation of NK cells prepared by the original process (Old) and NK cells proliferated by the re-stimulation process (New) was similar to, whereas NKG2C and 2B4 did not change after culture proliferation by both methods (FIG. 6). The expression of inhibitory receptors, CD158a and CD158e, remained largely unchanged upon incubation by both procedures (Old and New), whereas the ratios of NKG2A and CD158b were significantly increased ( FIGS. 12A-B ). All inhibitory receptor expression levels analyzed were similar in NK cells prepared by both methods. Expression of chemokine receptors such as CXCR3 and CXCR4 was also evaluated.

The frequency of CXCR3+ NK cells was significantly increased during NK cell proliferation by both methods (Old and New) with similar expression levels, whereas the frequency of CXCR4+ NK cells decreased after culture proliferation by both methods, and the new method ( new method) was slightly more decreased in NK cells prepared by (Fig. 12A-B). The expression level of CD62L was slightly increased in NK cells proliferated by both methods.

As can be seen from these results, the properties of IL21+ and IL21+/+ proliferated cells are similar, despite the fact that one set underwent a re-proliferation procedure.

Example 7: IL-21 Concentration

For various IL-21 concentrations, isolated CD56+ cells were treated with 10% FBS, 500 IU/mL IL-2, 10% FBS, 500 IU/mL IL-2 in RPMI medium containing 10, 30, 50, and 100 ng/mL IL-21 of type 1 experimental design. Resuspended in initial NK cell culture medium containing 20 μg/mL gentamicin (IL21+ only, no re-stimulation).

Example 8: IL-21 Concentration

Experiments in type 1 were performed at different ratios at 1:10:10 (CD56+ cells: LCL: KL-1), and 1:20:20, 1:30:30 (IL21+ only, no re-stimulation performed). of feeder cells.

Example 9

This non-limiting example shows IL-21 that freeze-thaw enhances proliferation of CD56+ and CD3-/CD56+ NK cells.

(1) Preparation of CD56+ natural killer cells (NK cells) -1

First, blood PBMCs were isolated using a Ficoll density gradient (Ficoll-Hypaque density gradient method). PBMCs were further treated according to the following 1-1 or 1-2.

1-1. CD56+ cell isolation

Add MACS buffer (1x PBS + 0.5% HSA) to suspend PBMCs and add CD56 microbeads (Miltenyi Biotec) to obtain 1-20 μL per 1.0 x 10 ⁷ PBMCs and incubate at 2-8 °C for 5-30 min. did. After incubation, MACS buffer was added and mixed, and the mixture was centrifuged (600xg) to precipitate the cells. After centrifugation, the supernatant was removed and the cells were resuspended by addition of MACS buffer, and the cells were added to a MACS separator coupled to the column. MACS buffer was passed through the column to remove non-specific binding. The column was removed from the MACS separator, transferred to a 15 mL conical tube, and MACS buffer was added to separate the CD56+ cells attached to the column.

1-2. CD3-/CD56+ cell isolation

CD3-/CD56+ cells were isolated as follows. Add MACS buffer (1x PBS + 0.5% HSA) to suspend PBMCs and add CD3 microbeads (Miltenyi Biotec) to yield 1-20 μL per 1.0 x 10 ⁷ PBMCs and incubate at 2-8 °C for 5-30 min. did. After incubation, MACS buffer was added and mixed, and the mixture was centrifuged (600xg) to precipitate the cells. After centrifugation, the supernatant was removed and the cells were resuspended by addition of MACS buffer, and the cells were added to a MACS separator coupled to the column. CD3-cells were recovered by passing MACS buffer through the column.

MACS buffer (1x PBS + 0.5% HSA) was added to the recovered CD3-cells to resuspend the CD3-cells, and CD56 microbeads (Miltenyi Biotec) were added to obtain 1-20 μL per 1.0x10 ⁷ CD3-cells. and incubated at 2-8 °C for 5-30 minutes. After incubation, MACS buffer was added and mixed, and the mixture was centrifuged (600xg) to precipitate the cells. After centrifugation, the supernatant was removed and the cells were resuspended by addition of MACS buffer, and the cells were added to a MACS separator coupled to the column. MACS buffer was passed through the column to remove non-specific binding. The column was removed from the MACS separator, transferred to a 15 mL conical tube, and MACS buffer was added to separate the CD3-/CD56+ cells attached to the column.

1-3. Invitational culture

CD56 + cells or CD3-/CD56 + cells isolated from 1-1 and 1-2 in RPMI-1640 medium containing 10% FBS at 37 °C 5% CO ₂ , 500 IU/mL and 50, respectively Co-cultured in an incubator with feeder cells (Jurkat cells, and EBV-LCL cells) prepared in advance by 100 Gy irradiation in the presence of ng/mL concentrations of IL-2 and IL-21.

On day 6, cells were inoculated into a 350 mL standard bag at 1.0 x 10 ⁵ - 2.0 x 10 ⁶ /mL and cultured for 4 more days, and on day 10, cells were inoculated at 1.0 x 10 ⁵ - 2.0 x 10 ⁶ cells/mL. and put it in a 1L bag and incubated for 4 more days. At this time, the ratio (CD56+ cells or CD3-/CD56+ cells):(Jurkat cells):(EBV-LCL cells) during incubation is 1:30:30.

1-4. Secondary culture after freezing and thawing

On the 14th day of culture 1(1-3), the cultured cells were suspended in a solution containing 90% FBS and 10% DMSO, stored frozen at -192°C or lower, and thawed in constant temperature water at 37°C according to the culture schedule. did it

Then, RPMI-1640 containing 10% FBS with IL-2 and IL-21 added at concentrations of 500 IU/mL and 50 ng/mL, respectively, was applied to 100 Gy irradiated feeder cells (Jurkat). cells and EBV-LCL cells), and then co-cultured in a 37° C. 5% CO ₂ incubator.

On day 6 after thawing and incubation, cells were seeded at 1.0x10 ⁵ -2.0x10 ⁶ cells/mL in a 350 mL bag and incubated for an additional 4 days, and on day 10, 1.0 x10 ⁵ - 2.0x10 ⁶ cells in a 1L standard bag. The cells were inoculated at /mL and further cultured for 4 days.

To maintain the growth of cells in culture up to 14 days after thawing, cells were treated with 100 Gy irradiated feeder cells (Jurkat cells and EBV-LCL cells), 500 IU/mL and 50 ng/mL, respectively. co-cultured in the presence of IL-2 and IL-21 at a concentration of Cells were cultured in RPMI-1640 medium containing 10% FBS added in an incubator at 37° C. 5% CO ₂ .

On the 20th day after thawing, the cells were inoculated into a 1L bag at 1.0x10 ⁵ -2.0x10 ⁶ cells/mL, then incubated for 4 more days, and on the 24th day after thawing, the cells were inoculated into a 1L bag at 1.0x10 ⁵ -2.0x10 It was inoculated at ⁶ cells/mL and further cultured for 4 days.

Finally, on the 28th day of culture after thawing, the cells were inoculated into a 1L bag at 1.0x10 ⁵ -2.0x10 ⁶ cells/mL, and further cultured for 3-6 days. At this time, (CD56+ cells or CD3-/CD56+ cells):(Jurkat cells):(EBV-LCL cells) were cultured at a ratio of 1:30:30.

(2) Preparation of CD56+ Natural Killer Cells-2

Except for the step of adding cytokines in 1-4, natural killer cells were prepared in the same manner as in (1).

(3) Comparative example. Preparation of Natural Killer Cells Excluding Cytokine Processing Steps

Natural killer cells were prepared in the same manner as in (1), except for the step of adding cytokine (IL-21) in 1-3 and 1-4.

(4) Confirmation of NK cell proliferation ability

The proliferative capacity of NK cells cultured by the methods of (1) to (3) was measured. As can be seen in FIG. 13A , when cytokines were not treated during the primary culture {(IL-21 -/-); See (3) above}, it was found that sufficient amounts of NK cells for clinical application were not generated after the freezing and thawing process (FIG. 13A). On the other hand, when the cells were treated with cytokines {(IL-21 +/+), see (1) above), sufficient amounts of NK cells for clinical application were generated even after freezing and thawing, and these results were frozen and cytokine treatment after thawing. Without cytokine treatment after freezing and thawing {(IL-21 +/-); See (2) above}, NK cells proliferated like cytokine-treated cells after freezing and thawing (FIG. 13B).

Example 10

Further results of some embodiments of various propagation and freezing and re-freezing procedures are shown in FIGS. 14A and 14B . 14A shows the resulting PDL (population doubling level) and depicts some embodiments of different periods of time during proliferation. 14B shows the resulting multiplication fold for the example.

In this example, it is analyzed whether NK cells can be stimulated or proliferated several times after freezing one or more times, and whether proliferation of NK cells can be stopped and resumed, rather than simply maintained. NK cells were initially cultured for 14 days. NK cells were treated with IL-21 (50 ng/mL) and feeder cells twice at 3-day intervals during the first 6-day period (0-6 days).

After this first 14-day proliferation, cells were frozen for 90 days, then thawed and cultured for another 14 days. NK cells were re-treated with IL-21 (50 ng/mL) and feeder cells twice at 3-day intervals during the first 6-day period (days 0-6) during this second proliferation.

Cells were re-stimulated a third time with IL-21 (50 ng/mL) and feeder cells twice at 3-day intervals during the first 6-day period (Day 0-6) during this second proliferation for 18 days. Finally re-cultured.

Proliferation of NK cells was monitored for 46 days of culture. As shown in Figures 14A and 14B, when feeder cells and IL-21 were treated twice or more, even after freezing the cells, NK cells showed significant proliferation after each stimulation.

Terms

The foregoing description of exemplary embodiments has been presented for purposes of illustration and description only, and is not intended to be exhaustive or to limit the invention to the precise form disclosed. Many modifications and variations are possible in light of the above teachings. Various combinations or subcombinations of the specific features and aspects of the embodiments disclosed above can be made and are still considered to be within one or more of the inventions. Moreover, the disclosure herein of any particular feature, aspect, method, attribute, characteristic, quality, attribute, element, etc., relating to an embodiment may be used in all other embodiments described herein. Accordingly, it should be understood that various features and aspects of the disclosed embodiments may be combined or substituted for each other to form various modes of the disclosed invention. Accordingly, it is intended that the scope of the invention disclosed herein should not be limited by the specific disclosed embodiments set forth above. Moreover, the present invention is susceptible to various modifications and alternative forms, specific examples of which are shown in the drawings and described in detail herein. However, this invention is not to be limited to the specific forms or methods disclosed, on the contrary, the invention is intended to cover all modifications, equivalents and alternatives falling within the spirit and scope of the various embodiments described and the appended claims. should be understood as inclusive. Not all methods disclosed herein need to be performed in the order in which they are recited. Methods disclosed herein include specific actions taken by physicians; However, it may also include third-party instructions for such action, whether express or implied. The ranges disclosed herein also include any and all overlaps, sub-ranges, and combinations thereof.

The embodiments were chosen and described in order to explain the principles of the invention and its practical application, so that those skilled in the art can utilize the invention and various embodiments and activate various modifications as are suited to the particular use contemplated. Alternative embodiments will be apparent to those skilled in the art, wherein the invention is defined by the appended claims rather than by the foregoing description and the exemplary embodiments set forth herein.

Conditional language, such as "can," "could," "might," or "may," Unless otherwise understood within the context in which it is used, it is generally intended to convey that certain embodiments include certain functions, elements and/or steps, whereas other embodiments do not. Accordingly, such conditional language is not generally intended to mean that functions, elements, and/or steps are required in any way for one or more embodiments.

The terms “comprising”, “including”, “having” are synonymous and are used in an inclusive, open-ended manner, and include additional elements, functions, acts, operations, etc. do not exclude Also, since the term "or" is used in an inclusive (but not exclusive) sense, for example, when used to link a list of elements, the term "or" means any of the elements of the list. means one, some or all.

The ranges disclosed herein also include any and all overlaps, sub-ranges, and combinations thereof. In languages such as "up to," "at least," "greater than," "less than," "between," Included.

As used herein, terms such as "approximately," "about," and "substantially" refer to the preceding numbers as referring to the recited numbers (e.g., about 10% = 10%). includes, and still represents an amount close to the stated amount that performs a desired function or achieves a desired result. For example, the terms “approximately,” “about,” and “substantially” can mean an amount within 10%, within 5%, within 1%, within 0.1%, within 0.01% of a specified amount.

As used herein, the term “generally” refers to a value, amount, or characteristic that predominantly includes or tends to include a particular value, amount, or characteristic. For example, in certain embodiments, the term "generally uniform" means exactly less than 20%, less than 15%, less than 10%, less than 5%, less than 1%, less than 0.1%, and less than 0.01%. Represents a uniform value, amount, or property.

The ranges disclosed herein also include any and all overlaps, sub-ranges, and combinations thereof. In languages such as "up to," "at least," "greater than," "less than," "between," Included. Numbers preceded by terms such as "about" or "approximately" include the recited number. For example, “about 5.0 cm” includes “5.0 cm”.

Some embodiments have been described with reference to schematic diagrams. However, it should be understood that the schematic drawings are not drawn to scale. The distances are illustrative only and do not necessarily represent an exact relationship with the actual dimensions and layout of the illustrated device.

For purposes of the present disclosure, certain aspects, advantages, and novel features are described herein. It should be understood that not all of these advantages may necessarily be achieved in accordance with any particular embodiment. Thus, for example, one of ordinary skill in the art would appreciate that this disclosure may be implemented or carried out in a manner that achieves one advantage or group of advantages as taught herein without necessarily achieving other advantages that may be taught or suggested herein. you will realize that you can

Moreover, while exemplary embodiments have been described herein, any of them having equivalent elements, modifications, omissions, combinations (eg, aspects of aspects spanning various embodiments), adaptations and/or changes. The scope of all embodiments will be recognized by those skilled in the art based on the present disclosure. The limitations of the claims are to be construed broadly based on the language used in the claims, and are not limited to the embodiments described herein or during the filing of the application, which embodiments are to be considered non-exclusive. should be interpreted Moreover, the operations of the disclosed processes and methods may be modified in any way, including reordering operations and/or inserting additional operations and/or deleting operations. Therefore, the specification and examples are to be regarded as illustrative only, with the true scope and spirit being indicated by the full scope of the claims and their equivalents.

Claims (56)

A method of propagating natural killer cells in culture, comprising:
isolating CD56+ cells from the blood sample;
co-culturing the isolated CD56+ cells in the presence of IL-21 for a first period;
freezing the co-cultured CD56+ cells after the first period;
thawing the frozen CD56+ cells; and
co-culturing the thawed CD56+ cells in the presence of IL-21 for a second period. The method of claim 1 , further comprising storing the frozen CD56+ cells at a temperature of less than -100°C. 3. The method of claim 1 or 2, further comprising storing the frozen CD56+ cells for at least one day prior to thawing. The method according to any one of the preceding claims, wherein the isolated CD56+ cells are co-cultured for 13-16 days prior to freezing. The method according to any one of the preceding claims, wherein the isolated CD56+ cells are co-cultured with one or more feeder cells irradiated in the presence of IL-21. The method according to any one of the preceding claims, wherein the thawed CD56+ cells are co-cultured with one or more feeder cells irradiated in the presence of IL-21. 7. The method of claim 5 or 6, wherein said one or more feeder cells are irradiated Jurkat cells, irradiated Epstein-Barr virus transformed lymphocyte continuous cell line (EBV-LCL) cells, K562 cells and The method of one or more selected from the group consisting of PBMCs. 8. The method of any one of claims 5-7, wherein said CD56+ cells are co-cultured at a ratio of CD56+ cells to feeder cells of about 1:1-100. 9. The method of claim 8, wherein said CD56+ cells are co-cultured at a ratio of CD56+ cells to feeder cells of about 1:2. 9. The method of claim 8, wherein said CD56+ cells are co-cultured at a ratio of CD56+ cells to feeder cells of about 1:5 to 1:30. 9. The method of claim 8, wherein said CD56+ cells are co-cultured at a ratio of CD56+ cells to feeder cells of about 1:10. 9. The method of claim 8, wherein said CD56+ cells are co-cultured at a ratio of CD56+ cells to feeder cells of about 1:30. 9. The method of claim 8, wherein said CD56+ cells are co-cultured at a ratio of CD56+ cells to feeder cells of about 1:1-100. The method according to any one of the preceding claims, wherein said IL-21 is added at a concentration of 10-100 ng/mL during the first and/or second period. 14. The method according to any one of claims 1 to 13, wherein said IL-21 is added at a concentration of 20-80 ng/mL during the first and/or second period. 14. The method according to any one of claims 1 to 13, wherein said IL-21 is added at a concentration of 30-70 ng/mL during the first and/or second period. The method according to any one of the preceding claims, wherein said IL-21 is added at least once during the first and/or second period. A method of propagating natural killer cells in culture, comprising:
isolating CD56+ from the blood sample;
co-culturing CD56+ cells with one or more feeder cells in the presence of IL-21;
freezing the CD56+ cells;
thawing the frozen CD56+ cells; and
Proliferating the thawed CD56+ cells. 19. The method of claim 18, wherein said freezing step freezes the CD56+ cells at a temperature of less than -100 °C. 20. The method of claim 18 or 19, further comprising storing the frozen CD56+ cells for a period of more than one day and less than 10 years. 21. The method of any one of claims 18-20, wherein said CD56+ cells are co-cultured for 13-16 days prior to freezing. 22. The method according to any one of claims 18 to 21, wherein said one or more feeder cells are irradiated Jurkat cells, irradiated Epstein-Barr virus transformed lymphocyte continuous cell line (EBV-LCL) cells; One or more methods selected from the group consisting of K562 cells and PBMCs. 23. The method of any one of claims 18-22, wherein the CD56+ cells are co-cultured at a ratio of CD56+ cells to feeder cells of about 1:1-100. 24. The method according to any one of claims 18 to 23, wherein said IL-21 is added at a concentration of 10-100 ng/mL. 25. The method of any one of claims 18-24, wherein said IL-21 is added one or more times. A method of increasing the cytotoxicity of natural killer cells, comprising:
providing said natural killer cells;
freezing the natural killer cells;
thawing the frozen natural killer cells; and
co-culturing the thawed natural killer cells with one or more feeder cells in the presence of IL-21. 27. The method of claim 26, further comprising storing the frozen natural killer cells at a temperature of less than -100 °C. 28. The method of claim 26 or 27, further comprising storing the frozen natural killer cells for at least one day prior to thawing. 29. The method according to any one of claims 26 to 28, wherein said one or more feeder cells are irradiated Jurkat cells, irradiated Epstein-Barr virus transformed lymphocyte continuous cell line (EBV-LCL) cells; The method of claim 1 or more selected from the group consisting of K562 cells and PBMCs. 30. The method of any one of claims 26-29, wherein the thawed natural killer cells are co-cultured at a ratio of CD56+ cells to feeder cells of about 1:1-100. 31. The method of any one of claims 26-30, wherein said IL-21 is added at a concentration of 10-100 ng/mL. 32. The method of any one of claims 26-31, wherein said IL-21 is added one or more times. A method of treating a subject, comprising:
collecting CD56+ cells from the subject;
co-culturing CD56+ cells with one or more feeder cells in the presence of IL-21;
freezing the co-cultured CD56+ cells for at least one day;
thawing the frozen CD56+ cells;
propagating thawed CD56+ cells; and
administering the proliferated CD56+ cells to the subject, wherein the cytotoxicity of the cells obtained from the second proliferation is at least 80% of the cytotoxicity of the co-cultured CD56+ cells prior to freezing. 34. The method of claim 33, further comprising storing the frozen CD56+ cells at a temperature of less than -100 °C. 35. The method of claim 33 or 34, further comprising storing the frozen CD56+ cells for at least one day prior to thawing. 36. The method of any one of claims 33-35, wherein the isolated CD56+ cells are co-cultured for 13-16 days prior to freezing. 37. The method of any one of claims 33-36, wherein the step of propagating the thawed CD56+ cells comprises co-culturing the thawed CD56+ with one or more irradiated feeder cells in the presence of IL-21. how it is. 38. The method of any one of claims 33 to 37, wherein said one or more feeder cells are irradiated Jurkat cells, irradiated Epstein-Barr virus transformed lymphocyte continuous cell line (EBV-LCL) cells; The method of one or more selected from the group consisting of K562 cells and PBMCs. 39. The method of any one of claims 33-38, wherein said CD56+ cells are co-cultured at a ratio of CD56+ cells to feeder cells of about 1:1-100. 40. The method according to any one of claims 33 to 39, wherein said IL-21 is added at a concentration of 10-100 ng/mL during the first and/or second period. 41. The method of any one of claims 33-40, wherein said IL-21 is added at least once during the first and/or second period. A composition comprising:
An effective amount of CD56+ cells derived from peripheral blood mononuclear cells (PBMCs) from a patient, the CD56+ cells are prepared by:
isolating peripheral blood mononuclear cells (PBMCs) from the blood sample;
isolating CD56+ cells from PBMCs;
co-culturing CD56+ cells with one or more feeder cells in the presence of one or more cytokines;
freezing the CD56+ cells;
thawing the frozen CD56+ cells; and
co-culturing the thawed CD56+ cells with one or more feeder cells in the presence of one or more cytokines. A cell composition comprising:
an effective amount of CD56+ cells derived from peripheral blood mononuclear cells (PBMCs) from a patient;
IL-2; and
IL-21. A composition comprising:
a first population of CD56+ cells derived from peripheral blood mononuclear cells (PBMCs);
ice; and
IL-2
IL-21,
wherein, when thawed, the CD56+ cells are cytotoxic to at least 80% of the second population of CD56+ cells, wherein the second population of CD56+ cells is not frozen. A method of propagating natural killer cells in culture, comprising:
providing PBMCs;
co-culturing the PBMCs in the presence of IL-21 for a first period;
freezing the co-cultured PBMCs after the first period;
thawing frozen PBMCs; and
co-culturing the thawed PBMCs in the presence of IL-21 for a second period. 46. The method of claim 45, wherein the ratio of PBMCs to feeder cells is 1:0.5:0.5. 18. A method according to any one of claims 2 to 17, which is subject to claim 45, rather than to claim 1. 34. The method of claim 33, further comprising freezing in an injection-prepared solution. 35. The method of claim 33 or 34, wherein said cytotoxicity is compared to non-frozen proliferation (in the presence or absence of IL-21) and growth frozen but co-cultured with IL-21+ in a first step. Method, the mean cytotoxicity of the frozen proliferation is 97.7% of that of the non-frozen. 35. The method of claim 33 or 34, wherein said cytotoxicity comprises comparing non-frozen proliferation (with or without IL-21) to proliferation frozen but not co-cultured with IL-21+ in the first step. wherein the mean cytotoxicity of the frozen proliferation is 81.4% of that of the non-frozen. 35. The method of claim 33 or 34, wherein said IL-21 is added both before and after freezing, and wherein said average cytotoxicity is 114% of non-frozen proliferation. A composition comprising:
IL-2;
5-10% DMSO;
90-95% FBS; and
NK cells, optionally CD56+ cells. 53. The composition of claim 52, wherein the composition is a frozen solid. 53. The composition of claim 52, wherein said NK cells are at least 90% of the cell population of the composition. A composition comprising:
IL-2;
5-10% DMSO;
80-95 % Hartman solution;
1-10% human serum albumin; and
NK cells. 56. The composition of any one of claims 52-55, further comprising a CryoStor solution.

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